Protease Activated Receptor-1 (PAR1) Derived Cytoprotective Polypeptides and Related Methods

ABSTRACT

The present invention provides novel PAR 1 derived cytoprotective oligopeptides or polypeptides which typically contain at least the first 4 N-terminal residues that are substantially identical to the corresponding N-terminal residues of Met 1 -Arg 46  deleted human PAR 1 sequence. These cytoprotective oligopeptides or polypeptides are capable of activating PAR 1 and promoting PAR 1 cytoprotective signaling activities. The invention also provides engineered cells or transgenic non-human animals which harbor in their genome an altered PAR 1 gene that is resistant to cleavage at Arg 41  and/or Arg 46  residues. Additionally provided in the invention are methods of screening candidate compounds to identity additional cytoprotective compounds or cytoprotective proteases. The invention further provides therapeutic use or methods of employing a PAR 1 derived cytoprotective oligopeptide or polypeptide to treat conditions associated with tissue injuries or undesired apoptosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage fling in accordance with 35 U.S.C.§371 of International Patent Application No. PCT/US2012/000546, filedNov. 7, 2012, which claims benefit to U.S. Provisional PatentApplication Ser. No. 61/628,834, filed Nov. 7, 2011, each of which isincorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under HL087618 andHL052246 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

The protease-activated receptor 1 (PAR1) is a thrombin receptor whichbelongs to the class of G protein-coupled receptors (GCPR). PAR1 isexpressed in various tissues, e.g., endothelial cells, smooth musclescells, fibroblasts, neurons and human platelets. It is involved incellular responses associated with hemostasis, proliferation, and tissueinjury. PAR1 is known to be activated by proteases, notably by thrombin,by cleavage at Arg⁴¹ or by peptides that mimic the new N-terminuscreated when cleavage at Arg⁴¹ occurs. Such peptides are often referredto as Thrombin Receptor Activating Peptides (TRAP). Thrombin cleavage ofPAR1 or treatment of cells with TRAP are generally proinflammatory andcan often be deleterious to cells or animals.

Plasma Protein C is a serine protease zymogen and is known for its milddeficiency linked to venous thrombosis risk and severe deficiency linkedto neonatal purpura fulminans. Activated Protein C (APC) exerts bothanticoagulant activity via proteolytic inactivation of factors Va andVilla and cellular cytoprotective actions via direct initiation of cellsignaling. Based on studies of engineered APC mutants and the use ofgenetically modified mice, APC's cell signaling actions are thought todrive murine APC's mortality reduction in sepsis models, neuroprotectiveactions in brain injury models, and nephroprotective effects in kidneyinjury models. These actions in vivo are generally suggested to involvemultiple receptors (PAR1, endothelial protein C receptor (EPCR), PAR3,and CD11b), while in vitro studies implicate these receptors andpotentially also other receptors (apoER2, beta1 and beta3 integrins,S1P1, and the angiopoietin/Tie-2 axis) for APC's cellular effects.Crosstalk among these receptors may permit a timely integration ofAPC-induced signaling which ultimately determines APC's effects on aspecific cell and organ.

Modulation of PAR1-mediated signaling activities could have varioustherapeutic applications. There is a need in the art for better meansfor such therapeutic applications. The present invention addresses thisand other related needs.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides isolated polypeptides(including short oligopeptides and longer polypeptides) derived fromprotease activated receptor-1 (PAR1). These PAR1-derived cytoprotectivepolypeptides typically have at least the first 4 N-terminal residuesthat are substantially identical to the corresponding N-terminalresidues of Met¹-Arg⁴⁶ deleted human PAR1 sequence (SEQ ID NO:2), avariant sequence or an ortholog sequence. In some related embodiments,the invention provides isolated polynucleotides or nucleic acidmolecules that encode the PAR1 derived polypeptides disclosed herein, aswell as expression vectors harboring such polynucleotides.

The PAR1 derived cytoprotective polypeptides or oligopeptides typicallycomprise from about 4 amino acid residues to about 350 amino acidresidues in length. In some embodiments, the polypeptides (comprise fromabout 8 amino acid residues to about 60 amino acid residues in length.In some embodiments, the polypeptides have at least the first 6N-terminal residues that are substantially identical to thecorresponding N-terminal residues of SEQ ID NO:2. In some otherembodiments, the polypeptides have at least the first 8 N-terminalresidues that are substantially identical to the correspondingN-terminal residues of SEQ ID NO:2. In still some other embodiments, thepolypeptides have at least the first 20 N-terminal residues that are atleast 90% identical to the corresponding N-terminal residues of SEQ IDNO:2.

Some preferred cytoprotective polypeptides of the invention have thefirst 20 N-terminal residues as shown in SEQ ID NO:4(NPNDKYEPFWEDEEKNESGL). Some of these polypeptides consist essentiallyof an amino acid sequence shown in any one of SEQ ID NOs:3, 4, and14-20. Some other embodiments of the invention are directed to variantpolypeptides or peptidomimetics that are derived from the preferredcytoprotective polypeptides exemplified herein. In some theseembodiments, the variant polypeptides contain one or more conservativelysubstituted residues in the N-terminus relative to SEQ ID NO:2.

In another aspect, the invention provides modified or mutant proteaseactivated receptor-1 (PAR1) molecules or fragments thereof. Relative toan unmodified or wildtype PAR1 (e.g., SEQ ID NO:1), the modified ormutant PAR1 molecules or fragments thereof comprise a missense mutationat residue Arg⁴⁶. Some of the modified or mutant PAR1 polypeptide orfragment thereof comprises an Arg⁴⁶Gln missense mutation. In someembodiments, the modified or mutant PAR1 polypeptide or fragment thereofcan additionally contain a missense mutation at residue Arg⁴¹. Forexample, they can additionally harbor an Arg⁴¹Gln substitution. In somerelated embodiments, isolated polynucleotides or nucleic acid moleculesencoding such modified or mutant PAR1 polypeptides or fragments thereofare provided in the invention.

In another aspect, the invention provides engineered host cells ortransgenic non-human animals. These engineered host cells or transgenicnon-human animals contain in their genome an altered or mutant proteaseactivated receptor-1 (PAR1) gene. The altered or mutant PAR1 geneencodes a PAR1 molecule that is resistant to protease cleavage at Arg⁴⁶.

In still another aspect, the invention provides methods for identifyingagents with cytoprotective activities for endothelial cells or othercells. These methods typically entail (1) contacting a candidate agentwith a cell expressing a mutant PAR1 or a PAR1 fragment that isresistant to thrombin cleavage; (2) detecting cleavage at Arg⁴⁶ in themutant PAR1 or the PAR1 fragment or detecting a PAR1 mediatedcytoprotective signaling activity. In some of the methods, the employedmutant PAR1 or PAR1 fragment contains a point mutation at Arg⁴¹, e.g.,an Arg⁴¹Gln substitution. In some methods, the employed cell expressingthe mutant PAR1 or PAR1 fragment is an endothelial cell. In someembodiments, the candidate agent is contacted with the cell in vivo viaadministration to a transgenic non-human animal.

Some of the screening methods of the invention employ candidate agentsthat are peptides, peptidomimetics or analog compounds. For example, thecandidate agents to be screened in the methods can be variants, analogsor peptidomimetics derived from the polypeptide shown in SEQ ID NO:4. Insome other screening methods, the employed candidate agents areproteases or variants thereof. In some of the screening methods,cytoprotective activity of the candidate agents is examined bymonitoring their effect on promoting activation of the PI3K-Akt survivalpathway or inhibition of apoptosis.

In yet another aspect, the invention provides methods of promotingcytoprotective activity for endothelial cells. These methods involvecontacting the cells with a PAR1-derived cytoprotective polypeptidewhich has at least the first 4 N-terminal residues that aresubstantially identical to the corresponding N-terminal residues ofMet¹-Arg⁴⁶ deleted human PAR1 sequence (SEQ ID NO:2). Some of thesemethods employ a PAR1-derived cytoprotective polypeptide which has atleast the first 6 N-terminal amino acid residues that are at least 90%identical to the corresponding N-terminal residues of SEQ ID NO:2. Someof the methods are directed to providing cytoprotective activity toendothelial cells in vivo (i.e., cells present in a subject). Forexample, a PAR1 derived cytoprotective polypeptide of the invention canbe administered to a subject to reduce mortality from adult severesepsis or pediatric meningococcemia; to promote wound healing indiabetic ulcer; to treat injuries from ischemic stroke, neurotrauma, orother acute or chronic neurodegenerative conditions; to treat injuriesfrom cardiac ischemia/reperfusion, hepatic ischemia/reperfusion, renalischemia/reperfusion; to treat inflammatory lung injury orgastrointestinal injury; to treat flap necrosis in reconstructivesurgery; to prolong survival following Ebola infection; or to reduceinjury caused by radiation.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the human PAR1 protein sequence(SEQ ID NO:1). As disclosed herein, the N-terminal domain is susceptibleto proteolytic cleavage at Arg⁴¹ or at Arg⁴⁶ after which the newlygenerated N-terminus which contains a free alpha-NH2 moiety at residues42 or 47, respectively, that can act as a tethered-ligand which inducesreceptor activation resulting in the initiation and activation ofintracellular G protein-coupled signaling pathways. In addition, thefollowing sequence regions have been identified: three extracellularloops designated e1, e2 and e3, seven roughly parallel, transmembranehelices, and four intracellular domains designated i1, i2, i3 and i4 ofwhich the latter is anchored into the inner membrane leaflet via acysteine palmitoylation and which comprises the C-terminal end. Aminoacid numbering starts with 1 at the methionine from the initiationcodon. The mature PAR1 protein is likely to start at residue Ala²⁶.

FIG. 2 shows that Activated Protein C cleaves a synthetic PAR1 peptide(TR33-62) at Arg⁴⁶.

FIG. 3 shows that Activated Protein C cleaves PAR1 at Arg⁴⁶ ontransfected HEK-293 cells.

FIG. 4 shows that Activated Protein C cleaves PAR1 at Arg⁴⁶ on EA.hy.926endothelial cells.

FIG. 5 shows that APC cleaves PAR1 at Arg⁴⁶ on untransfected EA.hy.926endothelial cells expressing endogenous EPCR and PAR1.

FIG. 6 shows activation of cell signaling (ERK 1/2) by APC, thrombin andTR42-51 peptide (SEQ ID NO:8) and cytoprotective TR47-66 peptide (SEQ IDNO:4) on EA.hy.926 endothelial cells.

FIG. 7 shows preferential activation of Akt by the TR47-66 peptide (SEQID NO:4) versus preferential activation of ERK1/2 by the TR42-51 peptide(SEQ ID NO:8) on EA.hy.926 endothelial cells.

FIG. 8 shows that PAR1 derived cytoprotective polypeptide TR47-66 (SEQID NO:4) conveys anti-apoptotic effects on EA.hy.926 endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The present invention is predicated in part on the discoveries by thepresent inventors that, in addition to the known cleavage site (Arg⁴¹)in PAR1, Activated Protein C (APC) also cleaves PAR1 at a secondcleavage site not previously known or envisioned in the art.Importantly, it was found that cleavage of PAR1 at the second siteresults in distinctly different consequences than caused by cleavage atthe first site, and that this alternative cleavage distinguishes APC'sfrom thrombin's effects. As detailed in the Examples below, theinventors first observed that APC cleaved a synthetic PAR1 N-terminalpeptide (TR33-62; SEQ ID NO:13) at Arg⁴¹, which is the same cleavagesite by thrombin. It was then found that APC also cleaved the PAR1TR33-62 peptide at an additional site distal from Arg⁴¹. Proteolysis ofthe TR33-62 peptide with APC resulted in fragments corresponding toTR33-41 and TR42-62 similar to thrombin. But in contrast to thrombin, athird fragment was generated by APC and the TR42-62 fragment disappearedover time with the concomitant accumulation of a novel peptide.Incubation of thrombin-cleaved TR42-62 with APC resulted in proteolysisof TR42-62 and generation of the novel fragment of TR47-62, indicatingthe existence of a second APC cleavage site in PAR1 that was distinctand distal from Arg⁴¹. Isolation of the novel proteolytic fragments andtheir MALDI-TOF analysis identified Arg⁴⁶ as the second APC cleavagesite in the TR33-62 peptide or TR42-62 peptide.

Additionally, the inventors observed that, when cells containingwild-type EPCR were transfected with SEAP-PAR1 wild type and mutantconstructs, both thrombin and APC cleaved wt-PAR1. As anticipated,efficient cleavage by thrombin was observed for R46Q-PAR1 but notR41Q-PAR1 or R41Q/R46Q-PAR1. In contrast, APC readily cleaved bothR41Q-PAR1 and R46Q-PAR1 whereas cleavage of R41Q/R46Q-PAR1 by APC wasnegligible. APC mediated cleavage of R41Q-PAR1 and R46Q-PAR1 requiredthe presence of functional EPCR and was not supported by theAPC-binding-defective E86A-EPCR mutant. These results indicate that oncells Arg⁴⁶ in PAR1 can serve as a second cleavage site for APC. Sincethe new PAR1 N-terminus after proteolysis acts as a tethered ligand forreceptor activation, cleavage at Arg⁴¹ vs. Arg⁴⁶ could createstructurally distinct agonists, which explains the divergent patternsfor PAR1-mediated cytoprotective APC signaling vs. proinflammatorythrombin signaling.

The inventors further examined whether the APC-induced new PAR1N-terminus starting at Asn⁴⁷ could promote signaling. It was found thata synthetic peptide with the PAR1 47-66 N-terminal sequence (SEQ IDNO:4) (termed “NPND”, “TR47” or “TR47-66” peptide) increased Aktphosphorylation at Ser⁴⁷³ in endothelial cells, whereas neither acontrol scrambled sequence (47-66)-peptide (SEQ ID NO:9) (termed“scrTR47” peptide) nor a TRAP peptide (SEQ ID NO:8) had a similarremarkable effect on Akt phosphorylation. Moreover, the NPND-peptide,but neither the scrambled sequence-related peptide nor a TRAP peptide,inhibited staurosporine-induced endothelial cell apoptosis. Theinventors additionally demonstrated that TR47-66 inducedvascular-endothelial protective effects in vitro and in vivo. These datasuggest that the new N-terminus generated by APC's cleavage at Arg⁴⁶ inPAR1 generates a novel tethered ligand that can induce cytoprotectiveAPC-like but not thrombin-like signaling characteristics.

In accordance with these discoveries, the present invention providesnovel PAR1 derived cytoprotective peptides or polypeptides. Thesepolypeptides are capable of activating cytoprotective signalingactivities mediated by PAR1 as demonstrated by the TR47 peptide (SEQ IDNO:4) exemplified herein. The invention also provides engineered cellsor transgenic non-human animals which harbor in their genome an alteredPAR1 gene that is resistant to cleavage at one or both of the cleavagesites (i.e., Are and Arg⁴⁶ for human PAR1). Additionally provided in theinvention are methods of screening candidate compounds to identityadditional cytoprotective peptides or polypeptides, as well as screeningmethods for identifying proteases which are capable of activating thecytoprotective PAR1 signaling via cleaving PAR1 at the second cleavagesite. The invention further provides therapeutic uses or methods ofemploying a PAR1 derived cytoprotective polypeptide to treat conditionsassociated with injuries or undesired apoptosis.

Unless otherwise stated, the present invention can be performed usingstandard procedures, as described, for example in Methods in Enzymology,Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon,G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998). The following sections provide additional guidance forpracticing the compositions and methods of the present invention.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Oxford Dictionary of Biochemistry and MolecularBiology, Smith et al. (eds.), Oxford University Press (revised ed.,2000); Dictionary of Microbiology and Molecular Biology, Singleton etal. (Eds.), John Wiley & Sons (3PrdP ed., 2002); and A Dictionary ofBiology (Oxford Paperback Reference), Martin and Hine (Eds.), OxfordUniversity Press (4PthP ed., 2000). In addition, the followingdefinitions are provided to assist the reader in the practice of theinvention.

The singular terms “a,” “an,” and “the” include plural referents unlessthe context clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

As used herein, the term “amino acid” of a peptide refers to naturallyoccurring and synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. The PAR1 derived protectivepolypeptides of the invention encompass derivative or analogs which havebe modified with non-naturally coding amino acids.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” refer to avariant which has conservative amino acid substitutions, amino acidresidues replaced with other amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

As used herein, a “derivative” of a reference molecule (e.g., acytoprotective polypeptide disclosed herein) is a molecule that ischemically modified relative to the reference molecule whilesubstantially retaining the biological activity. The modification canbe, e.g., oligomerization or polymerization, modifications of amino acidresidues or peptide backbone, cross-linking, cyclization, conjugation,fusion to additional heterologous amino acid sequences, or othermodifications that substantially alter the stability; solubility, orother properties of the peptide.

The term “engineered cell” or “recombinant host cell” (or simply “hostcell”) refers to a cell into which a recombinant expression vector hasbeen introduced. It should be understood that such terms are intended torefer not only to the particular subject cell but to the progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.

The term “fragment” refers to any peptide or polypeptide having an aminoacid residue sequence shorter than that of a full-length polypeptidewhose amino acid residue sequence is described herein. An isolatedpeptide of PAR1 is shortened or truncated compared to its parentfull-length PAR1. Relative to a full length PAR1 sequence, the PAR1derived cytoprotective polypeptides of the invention typically haveN-terminus truncation at the conserved Arg⁴⁶-Asn⁴⁷ residues. Thesefragments can additionally contain C-terminus truncations (e.g.,truncations of up to 50, 100, 200, 300 or more C-terminal residues)and/or also internal deletions.

The term “isolated” means the protein is removed from its naturalsurroundings. However, some of the components found with it may continueto be with an “isolated” protein. Thus, an “isolated polypeptide” is notas it appears in nature but may be substantially less than 100% pureprotein.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl.Math. 2:482c, 1970; by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity methodof Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988; bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, Madison, Wis.); or by manual alignment and visual inspection(see, e.g., Brent et al., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms thatare suitable for determining percent sequence identity and sequencesimilarity are the BLAST and BLAST 2.0 algorithms, which are describedin Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul etal., J. Mol. Biol. 215:403-410, 1990, respectively.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

Unless otherwise specified, the terms “polypeptide” and “peptide” areused interchangeably herein (e.g., “PAR1 derived cytoprotectivepolypeptide” and “PAR1 derived cytoprotective peptide”) to refer to apolymer of amino acid residues. They encompass both short oligopeptides(e.g., peptides with less than about 25 residues) and longer polypeptidemolecules (e.g., polymers of more than about 25 or 30 amino acidresidues). Typically, the PAR1 derived cytoprotective peptides(oligopeptides) or polypeptides of the invention can comprise from about4 amino acid residues to about 350 or more amino acid residues inlength. In some embodiments, the peptides or polypeptides comprise fromabout 8 amino acid residues to about 60 amino acid residues in length.The PAR1 derived cytoprotective peptides or polypeptides of theinvention include naturally occurring amino acid polymers andnon-naturally occurring amino acid polymer, as well as amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid.Unless otherwise indicated, a particular polypeptide sequence alsoimplicitly encompasses conservatively modified variants thereof.

As used herein, the term “peptide mimetic” or “peptidomimetic” refers toa derivative compound of a reference peptide (e.g., a cytoprotectivepolypeptide disclosed herein) that biologically mimics the peptide'sfunctions. Typically, the peptidomimetic derivative of a PAR1 derivedcytoprotective polypeptide of the invention has at least 50%, at least75% or at least 90% of the cytoprotective activities (e.g., inhibitionof endothelial cell apoptosis) of the reference polypeptide.

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, it refers tothe functional relationship of a transcriptional regulatory sequence toa transcribed sequence. For example, a promoter or enhancer sequence isoperably linked to a coding sequence if it stimulates or modulates thetranscription of the coding sequence in an appropriate host cell orother expression system. Generally, promoter transcriptional regulatorysequences that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory sequences, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

As used herein, the term “orthologs” or “homologs” refers topolypeptides that share substantial sequence identity and have the sameor similar function from different species or organisms. For example,PAR1 from human, rabbit, rat, mouse and many other animal species areorthologs due to the similarities in their sequences and functions.

The phrase “signal transduction pathway” or “signaling activities”(e.g., the PAR1 mediated cytoprotective signaling) refers to at leastone biochemical reaction, but more commonly a series of biochemicalreactions, which result from interaction of a cell with a stimulatorycompound or agent. Thus, the interaction of a stimulatory compound(e.g., PAR1 derived peptide shown in SEQ ID NO:4) with a cell generatesa “signal” that is transmitted through the signal transduction pathway,ultimately resulting in a cellular response.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, and reptiles.Except when noted, the terms “patient” or “subject” are used hereininterchangeably.

The term “transgene” means a nucleic acid sequence (e.g., one encoding amutant PAR1 polypeptide of the invention) which has been introduced intoa cell. A transgene could be partly or entirely heterologous, i.e.,foreign, to the transgenic animal or cell into which it is introduced,or can be homologous to an endogenous gene of the transgenic animal orcell into which it is introduced. A transgene can also be present in acell in the form of an episome. A transgene can include one or moretranscriptional regulatory sequences and any other nucleic acid, such as5′ UTR sequences, 3′ UTR sequences, or introns, that may be necessaryfor optimal expression of a selected nucleic acid.

A “transgenic animal” refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA.

As used herein, the term “treat” or treatment” refers to administrationof compounds or agents to prevent or delay the onset of the symptoms,complications, or biochemical indicia of a disease or condition,alleviating the symptoms or arresting or inhibiting further developmentof the disease or condition, or disorder. Treatment may be prophylactic(to prevent or delay the onset of the condition, or to prevent themanifestation of clinical or subclinical symptoms thereof) ortherapeutic suppression or alleviation of symptoms after themanifestation of the condition. For the therapeutic applications of thepresent invention, treatment is intended to reduce or alleviate at leastone adverse effect or symptom in medical conditions that are associatedwith undesired cell death or tissue injuries. Examples of suchconditions are described herein.

As used herein, the term “variant” refers to a molecule (e.g., apolypeptide or polynucleotide) that contains a sequence that issubstantially identical to the sequence of a reference molecule. Forexample, the reference molecule can be an N terminally truncated PAR1polypeptide (e.g., human PAR1 fragment starting at Asn⁴⁷ as shown in SEQID NO:2) or a polynucleotide encoding the polypeptide. The referencemolecule can also be a PAR1 derived cytoprotective polypeptide disclosedherein or a polynucleotide encoding the cytoprotective polypeptide(e.g., TR47 as shown in SEQ ID NO:4). In some embodiments, the variantcan share at least 50%, at least 70%, at least 80%, at least 90, atleast 95% or more sequence identity with the reference molecule. In someother embodiments, the variant differs from the reference molecule byhaving one or more conservative amino acid substitutions. In some otherembodiments, a variant of a reference molecule (e.g., a cytoprotectivePAR1 polypeptide) has altered amino acid sequences (e.g., with one ormore conservative amino acid substitutions) but substantially retainsthe biological activity of the reference molecule (e.g., activating PAR1cytoprotective signaling). Conservative amino acid substitutions arewell known to one skilled in the art.

The term “vector” is intended to refer to a polynucleotide moleculecapable of transporting another polynucleotide to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”).

III. PAR-1 Derived Cytoprotective Polypeptides and Derivative Compounds

The invention provides cytoprotective polypeptides (including shortpeptides or oligopeptides) which are derived from PAR1. PAR1 sequencesfrom human and many other non-human species have all been delineated inthe art. For example, human PAR1 cDNA sequence, which has correspondingamino acid sequence shown in SEQ ID NO:1, was originally reported in Vuet al., Cell 64:1057-1068, 1991 with revisions noted in GenBankaccession number NM_001992.3. The cytoprotective peptides orpolypeptides of the invention are capable of activating PAR1-mediatedcytoprotective signaling as disclosed herein. PAR-1 mediatedcytoprotective signaling or signaling activity refers to anynon-anticoagulant protective cellular activities mediated by theAPC-PAR1 signaling pathway. These include activation of the PI3k-Aktsurvival pathway, inhibition of endothelial apoptosis (e.g., by blockingthe pro-apoptotic activity of p53 or by other mechanisms), secretion ofTNF-α by macrophages, reduction of cellular NFκB activation inendothelial cells, prevention of leukocyte adhesion to activatedendothelial cells, induction of stabilization of endothelial cellbarrier integrity via sphingosine-1 phosphate release or sphingosine-1phosphate receptor 1 (S1P1) activation. As demonstrated in the Examplesbelow, cytoprotective activities of the PAR1-derived polypeptides of theinvention (e.g., SEQ ID NO:4) are evidenced by inhibition of apoptosisand promotion of cell survival.

Typically, the PAR1-derived cytoprotective polypeptides of the inventionhave at least the first 4 or 5 N-terminal residues that aresubstantially identical to the corresponding N-terminal residues ofMet¹-Arg⁴⁶ deleted human PAR1 sequence (SEQ ID NO:2), variants (e.g.,Met¹-Arg⁴⁶ deleted human PAR1 sequence with conservative substitutions)or orthologs (e.g., non-human PAR1 sequences with similar deletions).They can contain at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 200,300 or more amino acid residues in length. Some of the polypeptidescomprise from about 4 amino acid residues to about 100 amino acidresidues. Some of the polypeptides comprise from about 6 amino acidresidues to about 50 amino acid residues. In some embodiments, thecytoprotective PAR1-derived polypeptide has at least the first 6N-terminal residues that are substantially identical to thecorresponding N-terminal residues of SEQ ID NO:2. In some otherembodiments, the cytoprotective polypeptide has at least the first 7, 8,9 or 10 N-terminal residues that are substantially identical to thecorresponding N-terminal residues of SEQ ID NO:2. In still some otherembodiments, the polypeptide has at least the first 10, 15, 20, 25, ormore N-terminal residues that are substantially identical to thecorresponding N-terminal residues of SEQ ID NO:2. In some preferredembodiments, the PAR1-derived cytoprotective peptides of the inventionhave at least the first 4, 5, 6, 7, 8, 9, 10, 15, 20 or more N-terminalresidues that are 100% identical to the corresponding N-terminalresidues of the truncated PAR1 sequence shown in SEQ ID NO:2. A fewspecific examples of the PAR 1-derived cytoprotective polypeptides ofthe invention are shown in SEQ ID NOs: 3 and 4, and also peptides NPNDKY(SEQ ID NO:14), NPNDKYEP (SEQ ID NO:15), NPNDKYEPFW (SEQ ID NO:16),NPNDKYEPFWED (SEQ ID NO:17), NPNDKYEPFWEDEE (SEQ ID NO:18),NPNDKYEPFWEDEEKN (SEQ ID NO:19), and NPNDKYEPFWEDEEKNES (SEQ ID NO:20).

As noted above, the PAR1-derived cytoprotective peptides or polypeptidesalso include peptides or polypeptides that are derived from variantsequences of the Met¹-Arg⁴⁶ deleted human PAR1 sequence or non-humanPAR1 sequences. Thus, some of the cytoprotective polypeptides or theinvention have at least the first 4 N-terminal residues which aresubstantially identical to the corresponding residues in the non-humanPAR1 sequences which start at the conserved Asn residue. Based onalignment of human PAR1 extracellular fragment Asn⁴⁷-Try¹⁰⁰ (SEQ IDNO:3) with corresponding sequences from other species, the inventorsobserved that the sequence is extremely conserved in primates and hasonly moderate variation in different animals. Examples include PAR1sequences from Rhesus monkey, white-tufted-ear marmoset, Northernwhite-cheeked gibbon, African savanna elephant, chimpanzee, dog, cattle,rat and mouse. In particular, it is known that Arg⁴⁶-Asn⁴⁷ residues ofhuman PAR1 are conserved in various other non-human PAR1 orthologs.Alignment of various PAR1 ortholog sequences and the presence of theconserved Arg-Asn residues corresponding to Arg⁴⁶-Asn⁴⁷ of human PAR1were also reported in the art, e.g., Soto et al., J. Biol. Chem.285:18781-93, 2010. Thus, unless otherwise noted, the conserved residuesbridging the second cleavage site in PAR1 as disclosed herein arereferred to as Arg⁴⁶ and Asn⁴⁷ regardless of which species the PAR1 geneis from. It is understood that the precise positions of these tworesidues in a given PAR1 molecule is not necessarily at positions 46 and47. Rather, the exact positions of these residues in a PAR1 polypeptidesequence can be easily determined by, e.g., sequence alignment. PAR1sequences from the various non-human animals which contain thisconserved cleavage site and a N-terminal sequence substantiallyidentical to the human PAR1 sequence can all be readily employed in thepractice of the present invention. Examples include, e.g., mouse PAR1(Accession No. NM_010169.3), rat PAR1 (Acc. No. NM_012950.2) and dogPAR1 (Acc. No. XM_546059.2).

Cytoprotective compounds of the invention also encompass variants,analogs, peptidomimetics or other derivative compounds that can begenerated from the PAR1 derived cytoprotective polypeptides exemplifiedherein (e.g., peptide TR47 as shown in SEQ ID NO:4). These derivativecompounds can be subject to the screening methods described below toidentify cytoprotective compounds with optimized activities. In someembodiments, the derivative compounds are modified versions of theexemplified peptides which are generated by conservative amino acidsubstitutions. For example, conservatively modified variants ofpolypeptide NPND KYEPFWEDEE KNESGL (SEQ ID NO:4) include polypeptidesNPNDRYEPFWEDEEKNESGL (SEQ ID NO:5), NPNDKYEPFWEEDEEKNESGL (SEQ ID NO:6)and NPNDRYEPFWEDEDKNESGL (SEQ ID NO:7). In some other embodiments, thederivative compounds are variants produced by non-conservativesubstitutions to the extent that that they substantially retain theactivities of those peptides. Modification to a cytoprotective PAR1peptide of the invention can be performed with standard techniquesroutinely practiced in the art (e.g., U.S. Patent Applications20080090760 and 20060286636).

In some embodiments, the analogs or derivative compounds of anexemplified PAR1-derived cytoprotective polypeptide of the invention(e.g., SEQ ID NO:4) can contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamate, and can include amino acids that are notlinked by polypeptide bonds. Similarly, they can also be cyclicpolypeptides and other conformationally constrained structures. Methodsfor modifying a polypeptide to generate analogs and derivatives are wellknown in the art, e.g., Roberts and Vellaccio, The Peptides: Analysis,Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341, AcademicPress, Inc., New York, N.Y. (1983); and Burger's Medicinal Chemistry andDrug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620, John Wiley &Sons Inc., New York, N.Y. (1995).

Some other derivative compounds of the exemplified PAR1-derivedcytoprotective polypeptides are peptidomimetics. Peptidomimetics basedon a PAR1-derived cytoprotective polypeptide (e.g., SEQ ID NO:4)substantially retain the activities of the reference polypeptide. Theyinclude chemically modified polypeptides, polypeptide-like moleculescontaining non-naturally occurring amino acids, peptoids and the like,have a structure substantially the same as the reference polypeptidesupon which the peptidomimetic is derived (see, for example, Burger'sMedicinal Chemistry and Drug Discovery, 1995, supra). For example, thepeptidomimetics can have one or more residues chemically derivatized byreaction of a functional side group. In addition to side groupderivatizations, a chemical derivative can have one or more backbonemodifications including alpha-amino substitutions such as N-methyl,N-ethyl, N-propyl and the like, and alpha-carbonyl substitutions such asthioester, thioamide, guanidino and the like. Typically, apeptidomimetic shows a considerable degree of structural identity whencompared to the reference polypeptide and exhibits characteristics whichare recognizable or known as being derived from related to the referencepolypeptide. Peptidomimetics include, for example, organic structureswhich exhibit similar properties such as charge and charge spacingcharacteristics of the reference polypeptide. Peptidomimetics also caninclude constrained structures so as to maintain optimal spacing andcharge interactions of the amino acid functional groups.

In some other embodiments, the PAR1-derived cytoprotective polypeptidesdescribed herein can be dimerized or multimerized by covalent attachmentto at least one linker moiety. For example, the peptides or polypeptidescan be conjugated with a C₁₋₁₂ linking moiety optionally terminated withone or two —NH—0 linkages and optionally substituted at one or moreavailable carbon atoms with a lower alkyl substituent. The PAR1 derivedpeptides described herein can be joined by other chemical bond linkages,such as linkages by disulfide bonds or by chemical bridges. In someother embodiments, the cytoprotective peptides described herein can belinked physically in tandem to form a polymer of PAR1-derived peptides.The peptides making up such a polymer can be spaced apart from eachother by a peptide linker. In some embodiments, molecular biologytechniques well known in the art can be used to create a polymer of PAR1peptides. In some embodiments, polyethylene glycol (PEG) may serve as alinker that dimerizes two peptide monomers. For example, a single PEGmoiety containing two reactive functional groups may be simultaneouslyattached to the N-termini of both peptide chains of a peptide dimer.These peptides are referred to herein as “PEGylated peptides.” In someembodiments, the peptide monomers of the invention may be oligomerizedusing the biotin/streptavidin system.

Methods for stabilizing peptides known in the art may be used with themethods and compositions described herein. For example, using D-aminoacids, using reduced amide bonds for the peptide backbone, and usingnon-peptide bonds to link the side chains, including, but not limitedto, pyrrolinone and sugar mimetics can each provide stabilization. Thedesign and synthesis of sugar scaffold peptide mimetics are described inthe art, e.g., Hirschmann et al., J. Med. Chem. 36, 2441-2448, 1996.Further, pyrrolinone-based peptide mimetics present the peptidepharmacophore on a stable background that has improved bioavailabilitycharacteristics (see, e.g., Smith et al., J. Am. Chem. Soc. 122,11037-11038, 2000).

In some embodiment, derivative compounds of the exemplifiedcytoprotective PAR1 polypeptides include modifications within thesequence, such as, modification by terminal-NH₂ acylation, e.g.,acetylation, or thioglycolic acid amidation, byterminal-carboxylamidation, e.g., with ammonia, methylamine, and thelike terminal modifications. One can also modify the amino and/orcarboxy termini of the polypeptides described herein. Terminalmodifications are useful to reduce susceptibility by proteinasedigestion, and therefore can serve to prolong half-life of thepolypeptides in solution, particularly in biological fluids whereproteases may be present. Amino terminus modifications includemethylation (e.g., —NHCH₃ or —N(CH₃)₂), acetylation (e.g., with aceticacid or a halogenated derivative thereof such as α-chloroacetic acid,α-bromoacetic acid, or α-iodoacetic acid), adding a benzyloxycarbonyl(Cbz) group, or blocking the amino terminus with any blocking groupcontaining a carboxylate functionality defined by RCOO— or sulfonylfunctionality defined by R—SO₂—, where R is selected from the groupconsisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, andsimilar groups. One can also incorporate a desamino acid at theN-terminus (so that there is no N-terminal amino group) to decreasesusceptibility to proteases or to restrict the conformation of thepeptide compound. In some embodiments, the N-terminus is acetylated withacetic acid or acetic anhydride.

Carboxy terminus modifications include replacing the free acid with acarboxamide group or forming a cyclic lactam at the carboxy terminus tointroduce structural constraints. One can also cyclize the peptidesdescribed herein, or incorporate a desamino or descarboxy residue at thetermini of the peptide, so that there is no terminal amino or carboxylgroup, to decrease susceptibility to proteases or to restrict theconformation of the peptide. Methods of circular peptide synthesis areknown in the art, for example, in U.S. Patent Application No.20090035814; and Muralidharan and Muir, Nat. Methods, 3:429-38, 2006.C-terminal functional groups of the peptides described herein includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

The PAR1 derived cytoprotective polypeptide compounds described hereinalso serve as structural models for non-peptidic compounds with similarbiological activity. There are a variety of techniques available forconstructing compounds with the same or similar desired biologicalactivity as the PAR1 peptides, but with more favorable activity than thePAR1 peptide with respect to solubility, stability, and susceptibilityto hydrolysis and proteolysis. See, e.g., Morgan and Gainor, Ann. Rep.Med. Chem. 24:243-252, 1989. These techniques include, but are notlimited to, replacing the peptide backbone with a backbone composed ofphosphonates, amidates, carbamates, sulfonamides, secondary amines, andN-methylamino acids.

IV. Synthesis of PAR1 Derived Cytoprotective Polypeptides and RelatedCompounds

The PAR1 derived cytoprotective polypeptides described herein, includingvariants and derivatives thereof, can be chemically synthesized andpurified by standard chemical or biochemical methods that are well knownin the art. Some of the methods for generating analog or derivativecompounds of the PAR1 derived cytoprotective polypeptides are describedabove. Other methods that may be employed for producing thecytoprotective polypeptides of the invention and their derivativecompounds, e.g., solid phase peptide synthesis, are discussed below. Forexample, the peptides can be synthesized using t-Boc(tert-butyloxycarbonyl) or FMOC (9-flourenylmethloxycarbonyl) protectiongroup described in the art. See, e.g., “Peptide synthesis andapplications” in Methods in molecular biology Vol. 298, Ed. by JohnHowl; “Chemistry of Peptide Synthesis” by N. Leo Benoiton, 2005, CRCPress, (ISBN-13: 978-1574444544); and “Chemical Approaches to theSynthesis of Peptides and Proteins” by P. Lloyd-Williams, et. al., 1997,CRC-Press, (ISBN-13: 978-0849391422), Methods in Enzymology, Volume 289:Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields(Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906);U.S. Pat. Nos. 4,965,343, and 5,849,954.

Solid phase peptide synthesis, developed by R. B. Merrifield, 1963, J.Am. Chem. Soc. 85 (14): 2149-2154, was a major breakthrough allowing forthe chemical synthesis of peptides and small proteins. An insolublepolymer support (resin) is used to anchor the peptide chain as eachadditional alpha-amino acid is attached. This polymer support isconstructed of 20-50 μm diameter particles which are chemically inert tothe reagents and solvents used in solid phase peptide synthesis. Theseparticles swell extensively in solvents, which makes the linker armsmore accessible. Organic linkers attached to the polymer supportactivate the resin sites and strengthen the bond between the alpha-aminoacid and the polymer support. Chloromethyl linkers, which were developedfirst, have been found to be unsatisfactory for longer peptides due to adecrease in step yields. The PAM (phenylacetamidomethyl) resin, becauseof the electron withdrawing power of the acid amide group on thephenylene ring, provides a much more stable bond than the classicalresin. Another alternative resin for peptides under typical peptidesynthesis conditions is the Wang resin. This resin is generally usedwith the FMOC labile protecting group.

A labile group protects the alpha-amino group of the amino acid. Thisgroup is easily removed after each coupling reaction so that the nextalpha-amino protected amino acid may be added. Typical labile protectinggroups include t-Boc (tert-butyloxycarbonyl) and FMOC. t-Boc is a verysatisfactory labile group which is stable at room temperature and easilyremoved with dilute solutions of trifluoroacetic acid (TFA) anddichloromethane. FMOC is a base labile protecting group which is easilyremoved by concentrated solutions of amines (usually 20-55% piperidinein N-methylpyrrolidone). When using FMOC alpha-amino acids, an acidlabile (or base stable) resin, such as an ether resin, is desired.

The stable blocking group protects the reactive functional group of anamino acid and prevents formation of complicated secondary chains. Thisblocking group must remain attached throughout the synthesis and may beremoved after completion of synthesis. When choosing a stable blockinggroup, the labile protecting group and the cleavage procedure to be usedshould be considered. After generation of the resin bound syntheticpeptide, the stable blocking groups are removed and the peptide iscleaved from the resin to produce a “free” peptide. In general, thestable blocking groups and organic linkers are labile to strong acidssuch as TFA. After the peptide is cleaved from the resin, the resin iswashed away and the peptide is extracted with ether to remove unwantedmaterials such as the scavengers used in the cleavage reaction. Thepeptide is then frozen and lyophilized to produce the solid peptide.This is generally then characterized by HPLC and MALDI before beingused. In addition, the peptide should be purified by HPLC to higherpurity before use.

Commercial peptide synthesizing machines are available for solid phasepeptide synthesis. For example, the Advanced Chemtech Model 396 MultiplePeptide Synthesizer and an Applied Biosystems Model 432A Peptidesynthesizer are suitable. There are commercial companies that makecustom synthetic peptides to order, e.g., Abbiotec, Abgent, AnaSpecGlobal Peptide Services, LLC., Invitrogen, and rPeptide, LLC.

The PAR1 derived cytoprotective polypeptides and derivatives thereof canalso be synthesized and purified by molecular methods that are wellknown in the art. Recombinant polypeptides may be expressed in bacteria,mammal, insect, yeast, or plant cells. For example, conventionalpolymerase chain reaction (PCR) cloning techniques can be used to clonea polynucleotide encoding a PAR1 peptide or polypeptide, using the fulllength PAR1 cDNA sequence as the template for PCR Cloning.Alternatively, the sense and anti-sense strand of the coding nucleicacid can be made synthetically and then annealed together to form thedouble-stranded coding nucleic acid. Ideally, restriction enzymedigestion recognition sites should be designed at the ends of the senseand anti-sense strand to facilitate ligation into a cloning vector orother vectors. Alternatively, a 3′A-overhang can be include for thepurpose of TA-cloning that is well known in the art. Such coding nucleicacids with 3′A-overhangs can be easily ligated into the Invitrogentopoisomerase-assisted TA vectors such as pCR®-TOPO, pCR®-Blunt II-TOPO,pENTR/D-TOPO®, and pENTR/SD/D-TOPO®. The coding nucleic acid can becloned into a general purpose cloning vector such as pUC19, pBR322,pBluescript vectors (Stratagene Inc.) or pCR TOPO® from Invitrogen Inc.The resultant recombinant vector carrying the polynucleotide encoding aPAR1 peptide can then be used for further molecular biologicalmanipulations such as site-directed mutagenesis for variant PAR1 peptideand/or to reduce the immunogenic properties of the peptide or improveprotein expression in heterologous expression systems, or can besubcloned into protein expression vectors or viral vectors for thesynthesis of fusion protein comprising PAR1 peptides and proteinsynthesis in a variety of protein expression systems using host cellsselected from the group consisting of mammalian cell lines, insect celllines, yeast, bacteria, and plant cells.

In some related embodiments, the invention provides isolated orsubstantially purified polynucleotides (DNA or RNA) which encode thePAR1-derived cytoprotective polypeptides described herein. Expressionvectors and engineered host cells harboring the vectors for expressingpolynucleotides encoding the polypeptides are also provided in theinvention. The polynucleotide encoding a PAR1-derived cytoprotectivepolypeptide (e.g., peptide shown in SEQ ID NO:4) are operationallylinked to a promoter in the expression vectors. The expression constructcan further comprise a secretory sequence to assist purification of thepeptide from the cell culture medium. The host cells to which thevectors are introduced can be any of a variety of expression host cellswell known in the art, e.g., bacteria (e.g., E. coli), yeast cell, ormammalian cells.

Recombinant protein expression in different host cells can beconstitutive or inducible with inducers such as copper sulfate, orsugars such as galactose, methanol, methylamine, thiamine, tetracycline,or IPTG. After the protein is expressed in the host cells, the hostcells are lysed to liberate the expressed protein for purification. Apreferred purification method is affinity chromatography such asion-metal affinity chromatograph using nickel, cobalt, or zinc affinityresins for histidine-tagged PAR1 peptide. Methods of purifyinghistidine-tagged recombinant proteins are described by Clontech usingtheir Talon® cobalt resin and by Novagen in their pET system manual,10th edition. Another preferred purification strategy is byimmuno-affinity chromatography, for example, anti-Myc antibodyconjugated resin can be used to affinity purify Myc-tagged PAR1 peptide.Enzymatic digestion with serine proteases such as thrombin andenterokinase cleave and release the PAR1 peptide from the histidine orMyc tag, releasing the recombinant PAR1 peptide from the affinity resinwhile the histidine-tags and Myc-tags are left attached to the affinityresin.

Cell-free expression systems can also be used for producingcytoprotective PAR1 polypeptides of the invention. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. An example of a cell-free translation systemcapable of producing proteins in high yield is described by Spirin et.al., Science 242:1162, 1988. The method uses a continuous flow design ofthe feeding buffer which contains amino acids, adenosine triphosphate(ATP), and guanosine triphosphate (GTP) throughout the reaction mixtureand a continuous removal of the translated polypeptide product. Thesystem uses E. coli lysate to provide the cell-free continuous feedingbuffer. This continuous flow system is compatible with both prokaryoticand eukaryotic expression vectors. An example of large scale cell-freeprotein production is described in Chang et. al., Science 310:1950-3,2005.

Other commercially available cell-free expression systems include theExpressway™ Cell-Free Expression Systems (Invitrogen) which utilize anE. coli-based in-vitro system for efficient, coupled transcription andtranslation reactions to produce up to milligram quantities of activerecombinant protein in a tube reaction format; the Rapid TranslationSystem (RTS) (Roche Applied Science) which also uses an E. coli-basedin-vitro system; and the TNT Coupled Reticulocyte Lysate Systems(Promega) which uses a rabbit reticulocyte-based in-vitro system.

V. PAR1 with Altered Protease Cleavage Sites and Related Cells orNon-Human Animals

The present invention provides modified or mutant protease activatedreceptor-1 (PAR1) molecules or fragments thereof which have alteredprotease cleavage sites. Relative to an unmodified or wildtype PAR1(e.g., SEQ ID NO:1), the modified or mutant protease activatedreceptor-1 (PAR1) molecules or fragments thereof are resistant toprotease (e.g., APC) cleavage at the first cleavage site (i.e., Arg⁴¹),at the second cleavage site (i.e., Arg⁴⁶), or at both cleavage sites. Asexemplified in the Examples below, these modified PAR1 molecules orfragments typically contain a missense substitution at one or both ofthe cleavage sites. For example, some of the modified or mutant PAR1molecules or fragments thereof contain an Arg⁴⁶Gln missense mutation. Insome other embodiments, the modified or mutant PAR1 molecules orfragments thereof contain an Arg⁴¹Gln missense mutation. In still someother embodiments, the modified or mutant PAR1 molecules or fragmentsthereof contain both Arg⁴¹Gln and Arg⁴⁶Gln substitutions. Other examplesinclude Ala substitutions at positions 41 and/or 46. In relatedembodiments, isolated polynucleotides or nucleic acid molecules encodingsuch modified or mutant PAR1 polypeptides or fragments thereof (e.g., amodified PAR1 gene described below), as well as vectors harboring suchpolynucleotides are also provided in the invention. The modified PAR1molecules or fragments of the invention with altered protease cleavagesites, the related polynucleotides and vectors can all be generated withroutinely practiced techniques of biochemistry and molecular biologydescribed herein or the specific procedures detailed in the Examplesbelow.

The invention further provides engineered cells and transgenic non-humananimals which contain a modified PAR1 gene in the genome. Typically, thePAR1 gene in the genome of the engineered cells or transgenic animalscontains mutations that result in resistance to cleavage by proteasessuch as thrombin or APC. In some embodiments, the mutation renders theencoded PAR1 resistant to thrombin cleavage at the first cleavage site(i.e., cleavage at Arg⁴¹) but not resistant to APC cleavage at thesecond cleavage site (i.e., Arg⁴⁶). For example, the altered PAR1 genecan harbor a missense mutation at the codon for Arg⁴¹, e.g., Arg⁴¹Glnmutation. In some other embodiments, the modified PAR1 gene in thegenome of the cells or animals is resistant to APC cleavage. Forexample, the PAR1 gene can have missense mutations at both Arg⁴¹ andArg⁴⁶. In still some other embodiments, the engineered cells ortransgenic animals can harbor a modified PAR1 gene which encodes a PAR1protein that is resistance to cleavage at Arg⁴⁶ but not at Arg⁴¹.

The engineered cells harboring a mutant PAR1 gene described above can begenerated from any cell suitable for expressing a PAR polypeptide.Preferably, the engineered cells are generated with a cell whichnaturally expresses PAR1 (e.g., an endothelial cell as exemplified inthe Examples below). The transgenic non-human animals which harbor amutant PAR1 gene described above can be generated from any animalsuitable for transgene expression (e.g., mouse or rat). In somepreferred embodiments, the transgenic non-human animals are transgenicmice. The engineered host cells and transgenic non-human animals whichharbor a mutant PAR1 gene described above include cells or animals withmutations in the endogenous PAR1 gene, as well as cells or animals whichcontain an exogenous PAR1 mutant gene described herein (e.g., a mutanthuman PAR1 gene). Non-human animals that are either homozygous orheterozygous for the mutation in the PAR1 gene are included in theinvention. Preferably, the animal is homozygous for the mutation.

The engineered host cells or non-human animals harboring a mutant PAR1gene of the present invention can be generated using routinely practicedmethods well known in the art or exemplified herein. Typically, they canbe obtained by genetic manipulation. In some embodiments of theinvention, non-human animals harboring a desired PAR1 mutant gene arecreated by genetic alteration of the wildtype PAR1 gene in a non-humananimal, e.g., by targeted mutagenesis. Such gene targeting methods havebeen well described in the art for generating non-human animals thatcontain specific gene mutations, e.g., U.S. Pat. No. 6,284,944. Briefly,the first step in producing a gene-targeted non-human mammal is toprepare a DNA targeting vector. The targeting vector is designed toreplace, via homologous recombination, part of the endogenous PAR1 genesequence of a non-human mammal, so as to introduce the desired mutation(e.g., Arg⁴⁶Gln substitution). The targeting vector is used to transfectnon-human mammalian cell, e.g., a pluripotent, murine embryo-derivedstem (“ES”) cell. In the cell, homologous recombination (i.e., thegene-targeting event) takes place between the targeting vector and thetarget gene. The mutant cell is then used to produce intact non-humanmammals (e.g., by aggregation of murine ES cells to mouse embryos) togenerate germ-line chimeras. The germline chimeras are used to producesiblings heterozygous for the mutated targeted gene. Finally,interbreeding of heterozygous siblings yields non-human mammals (e.g.,mice) homozygous for the mutated target gene.

Targeting vectors for the practice of this invention can be constructedusing materials, information and processes known in the art. A generaldescription of the targeting vector used in this invention follows. Atargeting vector or replacement vector for use in this invention has twoessential functions: (1) to integrate specifically (and stably) at theendogenous PAR1 target gene; and (2) to replace a portion of theendogenous PAR1 gene, thereby introducing the desired mutation aroundthe Arg⁴⁶ and/or Arg⁴¹ residues. Those two essential functions depend ontwo basic structural features of the targeting vector. The first basicstructural feature of the targeting vector is a pair of regions, knownas “arms of homology”, which are homologous to selected regions of theendogenous PAR1 gene or regions flanking the PAR1 gene. This homologycauses at least part of the targeting vector to integrate into thechromosome, replacing part or all of the PAR1 target gene, by homologousrecombination. The second basic structural feature of the targetingvector consists of the actual base changes (mutations) to be introducedinto the target gene. The mutation(s) to be introduced into the PAR1target gene must be located within the “arms of homology.”

Gene targeting, which affects the structure of a specific endogenousgene in a cell, is to be distinguished from other forms of stabletransformation, wherein integration of exogenous DNA for expression in atransformed cell is not site-specific, and thus does not predictablyaffect the structure of any particular gene already in the transformedcell. Furthermore, with the type of targeting vector preferred in thepractice of this invention, a reciprocal exchange of genomic DNA takesplace (between the “arms of homology” and the target gene), andchromosomal insertion of the entire vector is advantageously avoided.

Various targeting vectors can be employed to practice the presentinvention. For a given vector, the length of the arms of homology thatflank the replacement sequence can vary considerably without significanteffect on the practice of the invention. The arms of homology must be ofsufficient length four effective heteroduplex formation between onestrand of the targeting vector and one strand of a transfected cell'schromosome, at the PAR1 target gene locus. The base pairs to be changedin the PAR1 target gene must lie within the sequence that constitutesthe arms of homology. The arms of homology may lie within the PAR1target gene, but it is not necessary that they do so and they may flankthe PAR1 target gene.

Preferably, the targeting vector will comprise, between the arms ofhomology, a positive selection marker. The positive selection markershould be placed within an intron of the target gene, so that it will bespliced out of mRNA and avoid the expression of a target/marker fusionprotein. More preferably the targeting vector will comprise twoselection markers; a positive selection marker, located between the armsof homology, and a negative selection marker, located outside the armsof homology. The negative selection marker is a means of identifying andeliminating clones in which the targeting vector has been integratedinto the genome by random insertion instead of by homologousrecombination. Exemplary positive selection markers are neomycinphosphotransferase and hygromycin β phosphotransferase genes. Exemplarynegative selection markers are Herpes simplex thymidine kinase anddiphtheria toxin genes.

To eliminate potential interference on expression of the target protein,the positive selection marker can be flanked by short loxP recombinationsites isolated from bacteriophage P1 DNA. Recombination between the twoloxP sites at the targeted gene locus can be induced by introduction ofcre recombinase to the cells. This results in the elimination of thepositive selection marker, leaving only one of the two short loxP sites(see, e.g., U.S. Pat. No. 4,959,317). Excision of the positiveselectable marker from the mutated PAR1 gene can thus be effected.

If base pair changes (mutations) are introduced into one of the arms ofhomology, it is possible for these changes to be incorporated into thecellular gene as a result of homologous recombination. Whether or notthe mutations are incorporated into cellular DNA as a result ofhomologous recombination depends on where the crossover event takesplace in the arm of homology bearing the changes. For example, thecrossover in the arm occurs proximal to the mutations and so they arenot incorporated into cellular DNA. In another scenario, the crossovertakes place distal to the position of the mutations and they areincorporated into cellular DNA. Because the location of the crossoverevent is random, the frequency of homologous recombination events thatinclude the mutations is increased if they are placed closer to thepositive selection marker.

By the above method, the skilled artisan can achieve the incorporationof the selectable marker at a preselected location in the PAR1 targetgene flanked by specific base pair changes. Presumably, the artisanwould preferably choose to have the selectable marker incorporatedwithin the intron of the target gene so as not to interfere withendogenous gene expression while the mutations would be included inadjacent coding sequence so as to make desired changes in the proteinproduct of interest. See Askew et al., Mol. Cell. Biol. 13: 4115-4124,1993; Fiering et al., Proc. Natl. Acad. Sci. USA 90: 8469-8473, 1993;Rubinstein et al., Nuc. Acid Res. 21: 2613-2617, 1993; Gu et al., Cell73: 1155-1164, 1993; and Gu, et al., Science 265: 103-106, 1994).

In addition to non-human animals which have its endogenous PAR1 genemutated in accordance with the present invention, transgenic animalsharboring a heterologous PAR1 mutant gene are also included in thepresent invention. For example, a transgenic non-human animal of thepresent invention can be a mouse which contains a transgene whichencodes a human PAR1 mutant gene. Typically, such transgenic animalshave their endogenous PAR1 gene replaced with the PAR1 mutant transgene.Transgenic animals (e.g., transgenic mice) expressing a mutant PAR1 genefrom a different species (e.g., human) can be generated according tomethods well known in the art. For example, techniques routinely used tocreate and screen for transgenic animals have been described in, e.g.,see Bijvoet et al., Hum. Mol. Genet. 7:53-62, 1998; Moreadith et al., J.Mol. Med. 75:208-216, 1997; Tojo et al., Cytotechnology 19:161-165,1995; Mudgett et al., Methods Mol. Biol. 48:167-184, 1995; Longo et al.,Transgenic Res. 6:321-328, 1997; U.S. Pat. Nos. 5,616,491; 5,464,764;5,631,153; 5,487,992; 5,627,059; 5,272,071; and WO 91/09955, WO93/09222, WO 96/29411, WO 95/31560, and WO 91/12650. Methods forgenerating transgenic non-human animals expressing a mutant human geneare also taught in the art, e.g., U.S. Pat. No. 6,284,944.

VI. Screening Methods for Identifying Cytoprotective Agents andProteases

The novel PAR1-derived cytoprotective peptides and engineered cells ortransgenic animals expressing the modified PAR1 receptor can be used toidentify additional compounds and proteases with cytoprotectiveactivities. Some embodiments of the invention are directed to identifyadditional cytoprotective peptides or polypeptides based on the specificPAR1-derived cytoprotective peptides exemplified herein. For example,the TR47 peptide (SEQ ID NO:4) can be used as a scaffold to generate alibrary of variant or analog peptides and peptidomimetics. The libraryof candidate agents based on a reference—polypeptide (e.g., TR47 peptideshown in SEQ ID NO:4) can be readily produced using routinely practicedmethods as described herein. Such a library of candidate agents can thenbe screened for optimized or improved cytoprotective activities relativeto the activities of the reference polypeptide. The candidate agents canbe screened for improvement in any of the cytoprotective activities ofthe reference polypeptide disclosed herein, e.g., activation of thePI3k-Akt survival pathway or inhibition of staurosporine-inducedendothelial cell apoptosis (see Examples below and also Mosnier et al.,Biochem J. 373:65-70, 2003). Variants or analog compounds based on areference PAR1-derived cytoprotective peptide (e.g., TR47) which areoptimized with such a screening method can be employed in the varioustherapeutic applications described herein.

In some other embodiments, the invention provides screen methods foridentifying proteases which can exert cytoprotective activities viasignaling through PAR1 (e.g., activating PAR1 via cleavage at the secondcleavage site, Arg⁴⁶). Typically, candidate proteases agents arescreened for ability to cleavage the mutant PAR1 in vitro at Arg⁴⁶and/or elicit a cytoprotective cell signaling in vivo. The screeningmethods can employ the various assays for determining PAR1 cleavage andfor monitoring PAR1 mediated cytoprotective signaling, which areexemplified herein and also well known in the art (see Examples below).In some embodiments, the candidate agents can be screened for activityin cleaving a PAR1 N-terminal peptide harboring the Arg⁴⁶ residue (e.g.,the peptide of SEQ ID NO:13) via reverse phase HPLC as described inExample 1 below. In some other embodiments, cleavage at Arg⁴⁶ bycandidate agents can be examined with a PAR1 cleavage reporter constructwhich expresses in a host cell (e.g., HEK-293 cells) a labeled PAR1reporter molecule, e.g., PAR1 with a secreted alkaline phosphatase(SEAP) marker fused to its N-terminus as described in Example 2. Hostcells stably expressing the reporter PAR-1 molecule are used to screenfor candidate agents that can cleave PAR1 at Arg⁴⁶ and release thedetectable SEAP marker.

Alternatively or additionally, the candidate protease agents arescreened for ability to activate or promote a cytoprotective signalingactivity mediated by PAR1. In these embodiments, an engineered cell(e.g., an endothelial cell) or transgenic non-human animal (e.g., amouse) which contains a mutation in the PAR1 gene that renders theencoded PAR1 resistant to thrombin cleavage (e.g., human PAR1 withArg⁴¹Gln mutation) is employed. Candidate agents are contacted with thecell or administered to the animal to determine whether they are able toelicit a cytoprotective signaling activity in the cell or the animal.For example, they can be tested for ability to promote phosphorylationof Akt or ERK1/2 in host cells (e.g., EA.hy.926 endothelial cells) asdescribed in Examples 4 and 5 below. Ability of the candidate agent topromote PAR1 cytoprotective signaling can also be assessed by monitoringanti-apoptotic effects in host cells expressing the modified PAR1 genedescribed herein (e.g., PAR1 with Arg⁴¹Gln mutation). Example of such anassay scheme is described in Example 6 below which examined PAR1-derivedcytoprotective polypeptides for inhibiting staurosporine-inducedendothelial cell apoptosis in EA.hy.926 endothelial cells. An ability tocleave PAR1 at Arg⁴⁶ and/or to elicit a PAR1 mediated cytoprotectivesignaling activity in cells or animals (e.g., cells expressing a PAR1Arg⁴¹Gln mutant) would indicate that the identified agents arecytoprotective proteases.

For identifying proteases which selectively promote cytoprotective PAR1signaling, the candidate proteases obtained from the initial screeningcan be subject to an additional screening step. In this additional step,the candidate agents or proteases are screened for the lack of activityin cleaving the first cleavage site in PAR1 (e.g., Arg⁴¹ in human PAR1)and/or in activating proinflammatory thrombin signaling. In somemethods, this additional screening step is performed with an engineeredcell or animal that expresses a mutant PAR1 that is resistant tocleavage at the second cleavage site (e.g., human PAR1 with Arg⁴⁶Glnmutation). Cleavage of PAR1 at the first cleavage site and activation ofproinflammatory thrombin signaling can be monitored with the standardtechniques described herein or well known in the art. For example,activation of proinflammatory thrombin signaling can be examined via theroutinely practiced assay for phospholipid exposure on platelets andrelated phospholipid-dependent coagulation assay (see, e.g., Anderson etal., Proc. Natl. Acad. Sci. 96: 11189-93, 1999) or determining theability of the proteases to stimulate calcium ion fluxes in cells or theability to phosphorylate ERK1/2 in cells. This additional step allowsidentification of proteases that are capable of selectively activatingthe cytoprotective PAR1 signaling but not the thrombin-likeproinflammatory PAR1 signaling.

To ensure that the observed cytoprotective signaling is mediated throughPAR1, the screening methods can optionally further include a controlstep by examining the candidate proteases' activity in the presence of aPAR1 inhibitor. For example, small molecule selective antagonists ofPAR1 such as SCH 79797 can be used in the screening. SCH79797 and otherPAR1 antagonists are well known and readily available to the skilledartisans. See, e.g., Ahn et al., Biochem. Pharmacol. 60: 1425-1434,2000; Lidington et al., Am. J. Physiol., Cell Physiol. 289: C1437-C1447,2005; and Damiano et al., Cardiovasc. Drug Rev. 21: 313-26, 2003. Asanother optional control step, the identified proteases can also beexamined for cytoprotective activities in an engineered cell or animalthat expresses a mutant PAR1 that is resistant to cleavage at bothcleavage sites (e.g., human PAR1 with Arg⁴¹Gln/Arg⁴⁶Gln doublemutations). Failure to cleave such a double PAR1 mutant and/or to elicitany cytoprotective signaling response indicates that the observedcytoprotective activities via the Arg⁴¹Gln PAR1 mutant is mediatedthrough PAR1.

Other than screening for proteases which activate the cytoprotectivePAR1 signaling pathway, the screening methods of the invention can alsobe employed to identify other types of cytoprotective agonist compoundsof PAR1. In these methods, the candidate agents are typically screenedfor ability in activating PAR1 mediated cytoprotective signaling, e.g.,activating the PI3k-Akt survival pathway or inhibition ofstaurosporine-induced endothelial cell apoptosis as described herein.Similar to the above described screening methods for identifyingcytoprotective proteases, cells and/or animals expressing a PAR1 mutantresistant to cleavage at the first cleavage site (Arg⁴¹) may beemployed. Similarly, PAR1 bearing mutation at the second cleavage siteor at both the first and the second cleavage sites may be employed incontrol screening steps.

To identify proteases with cytoprotective activities, the candidatecompounds to be employed in the screening can be any naturally existingor recombinantly produced polypeptides or enzymes (including knownproteases). Preferably, the candidate agents or compounds used to screenfor novel cytoprotective proteases are proteases, variants or analogs.For example, the candidate agents subject to the screening methods canbe, e.g., any serine proteases, metallo proteinases, plasma proteases,cell membrane proteases, cell proteases, engineered proteases, and etc.Many specific examples of proteases from these enzyme classes are allwell known in the art and can be readily obtained commercially orrecombinantly produced. Other candidate proteases and specific examplesof human or mouse proteases suitable for the screening methods of theinvention are described in the art, e.g., Overall et al., Nat. Rev. Mol.Cell. Biol. 8:245-57, 2007; Doucet et al., Mol. Aspects Med. 29:339-58,2008; and Puente et al., Nat. Rev. Genet. 4:544-58, 2003. In some otherembodiments, the candidate agents employed for identifying novelcytoprotective proteases can be variants of a known protease. Forexample, variants or analogs of Activated Protein C (APC), variants ofthe active forms of prothrombin (active enzyme thrombin), coagulationfactor VII (active enzyme factor Vila), coagulation factor X (activeenzyme factor Xa) or variants or matriptase can all be used as candidateagents in the screening methods of the invention. Variants of theseproteases can be generated using routinely practiced chemical orbiochemical techniques as described herein or well known in the art. Forexample, APC variants that can be used to screen for novelcytoprotective proteases in the methods of the invention are describedin U.S. Patent Application 20100028910.

For identifying PAR1 agonist compounds, the candidate agents to bescreened with methods of the invention can be derivative or mimeticcompounds of the PAR1 derived cytoprotective polypeptides exemplifiedherein. For examples, the candidate agents can be polypeptides derivedfrom the polypeptide of SEQ ID NO:2 with various C-terminal truncations(e.g., polypeptides of SEQ ID NOs: 3, 4, and 14-20) or internaldeletions. The candidate agents can also be a library of polypeptidesderived from the polypeptide of SEQ ID NO:4 which contain one or moreamino acid substitutions. Methods for preparing libraries containingdiverse populations of peptides, peptoids and peptidomimetics are wellknown in the art and various libraries are commercially available. See,e.g., Ecker and Crooke, Biotechnology 13:351-360, 1995; and Blondelle etal., Trends Anal. Chem. 14:83-92, 1995; and the references citedtherein. See, also, Goodman and Ro, Peptidomimetics for Drug Design, in“Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E.Wolff; John Wiley & Sons 1995), pages 803-861; and Gordon et al., J.Med. Chem. 37:1385-1401 (1994). One skilled in the art understands thata peptide can be produced in vitro directly or can be expressed from anucleic acid, which can be produced in vitro. A library of peptidemolecules also can be produced, for example, by constructing a cDNAexpression library from mRNA collected from a tissue of interest.Methods for producing such libraries are well known in the art (see, forexample, Sambrook et. al., Molecular Cloning: A laboratory manual (ColdSpring Harbor Laboratory Press 1989).

Methods of designing peptide derivatives and mimetics and screening offunctional peptide mimetics are well known to those skilled in the art.One basic method of designing a molecule which mimics a known protein orpeptide is first to identify the active region(s) of the known protein(for example, in the case of an antibody-antigen interaction, oneidentifies which region(s) of the antibody that permit binding to theantigen), and then searches for a mimetic which emulates the activeregion. Although the active region of a known polypeptide is relativelysmall, it is anticipated that a mimetic will be smaller (e.g., inmolecular weight) than the protein, and correspondingly easier andcheaper to synthesize and/or have benefits regarding stability or otheradvantageous pharmacokinetic aspects. Such a mimetic could be used as aconvenient substitute for the polypeptide (e.g., SEQ ID NO:4), as anagent for interacting with the target molecule (e.g., PAR1). Forexample, Reineke et al. (Nat. Biotech. 17; 271-275, 1999) designed amimic molecule which mimics a binding site of the interleukin-10 proteinusing a large library of short synthetic peptides, each of whichcorresponded to a short section of interleukin 10. The binding of eachof these peptides to the target (in this case an antibody againstinterleukin-10) was then tested individually by an assay technique, toidentify potentially relevant peptides. Phage display libraries ofpeptides and alanine scanning methods can be used.

Other methods for designing peptide mimetics to a particular peptide orprotein include those described in European Patent EP1206494, theSuperMimic program by Goede et. al., BMC Bioinformatics, 7:11, 2006; andMIMETIC program by Campbell et al., Microbiol. and Immunol. 46:211-215,2002. The SuperMimic program is designed to identify compounds thatmimic parts of a protein, or positions in proteins that are suitable forinserting mimetics. The application provides libraries that containpeptidomimetic building blocks on the one hand and protein structures onthe other. The search for promising peptidomimetic linkers for a givenpeptide is based on the superposition of the peptide with severalconformers of the mimetic. New synthetic elements or proteins can beimported and used for searching. The MIMETIC computer program, whichgenerates a series of peptides for interaction with a target peptidesequence, is taught by W. Campbell et. al., 2002. In depth discussion ofthe topic is reviewed in “Peptide Mimetic Design with the Aid ofComputational Chemistry” by James R. Damewood Jr. in Reviews inComputational Chemistry, January 2007, Volume 9, Editor(s): Kenny B.Lipkowitz, Donald B. Boyd (John Wiley & Sons, Inc.); and in Tselios, et.al., Amino Acids, 14: 333-341, 1998.

VII. Therapeutic Applications and Pharmaceutical Compositions

The cytoprotective compounds or agents of the invention, including thevarious PAR1-derived cytoprotective peptides, polypeptides,peptidomimetics, variants or analogs described herein, can be employedin many therapeutic or prophylactic applications by stimulating PAR1mediated cytoprotective signaling activities. These applications areintended to achieve a desired therapeutic effect such as inhibition ofapoptosis or cell death, promotion of cell survival, cytoprotection,neuroprotection, or combinations thereof. In therapeutic applications, acomposition comprising a cytoprotective polypeptide or peptidomimetic isto provide cytoprotection to cells at risk for undergoing apoptotic celldeath or stress-induced injury either in vivo or in vitro. Thecomposition contains a cytoprotective peptide or peptidomimetic in anamount sufficient to cure, partially arrest, or detectably slow theprogression of the cell death or injury. In prophylactic applications,compositions containing a cytoprotective peptide or peptidomimetic areused to prevent apoptotic cell death or injury to a subject who is atthe risk of, or has a predisposition, to developing a condition withundesired apoptotic cell death or injury. Such applications allow thesubject to enhance the subject's resistance to, or to retard theprogression of, the condition.

Typically, the cytoprotective compounds of the invention (e.g., PAR1polypeptides of SEQ ID NOs: 4-7 and 14-20) are formulated inpharmaceutical compositions for the therapeutic or prophylacticapplications disclosed herein. The therapeutic compositions may beadministered in vitro to cells in culture, in vivo to cells in the body,or ex vivo to cells outside of a subject, which may then be returned tothe body of the same subject or another. The cells may be removed from,transplanted into, or be present in the subject (e.g., geneticmodification of endothelial cells in vitro and then returning thosecells to brain endothelium).

Subject who are suitable for the therapeutic or prophylacticapplications of the present invention are those who could benefit fromPAR1 cytoprotective signaling activities. Such subjects include patientsat risk for damage to blood vessels or other tissue organs, which damageis caused at least in part by apoptosis. The risk for cell damage may bethe result of any one or more of sepsis, ischemia/reperfusion injury,stroke, ischemic stroke, acute myocardial infarction, acuteneurodegenerative disease, chronic neurodegenerative disease, organtransplantation, chemotherapy, or radiation injury. These causes of celldamage are not intended in any way to limit the scope of the invention,as one skilled in the art would understand that other diseases orinjuries also may put cells at risk for damage caused at least in partby apoptosis. The effective doses or therapeutic doses will be thosethat are found to be effective at preventing or alleviating cell damagecaused at least in part by apoptosis. In some embodiments, thecytoprotective compounds of the invention can be applied to cells ortissue in vitro or in situ. In some other embodiments, thecytoprotective compounds are administered to achieve a desiredtherapeutic effect in vivo in subjects afflicted with or at risk ofdeveloping a condition with associated with undesired cell death orinjury. Examples of specific conditions and desired therapeutic effectsinclude the following: reduction of mortality in sepsis (e.g., adultsevere sepsis); reduction of death in pediatric meningococcemia;promotion of diabetic ulcer wound healing; treat or prevent injuries inischemic stroke, neurotrauma, and other acute or chronicneurodegenerative conditions; treat or prevent injuries in cardiacischemia/reperfusion, hepatic ischemia/reperfusion, renalischemia/reperfusion; treat or prevent inflammatory lung injury,gastrointestinal injury; treat or prevent flap necrosis inreconstructive surgery; prolong survival following Ebola infection; andreduce damage to a subject caused by radiation.

Pharmaceutical compositions of the invention can be prepared inaccordance with methods well known and routinely practiced in the art.See, e.g., Remington: The Science and Practice of Pharmacy, MackPublishing Co., 20^(th) ed., 2000; and Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions. The pharmaceutical compositions of the inventionmay be administered by any known route. By way of example, thecomposition may be administered by a mucosal, pulmonary, topical, orother localized or systemic route (e.g., enteral and parenteral). Inparticular, achieving an effective amount of the cytoprotective compoundin the central nervous system may be desired. This may involve a depotinjection into or surgical implant within the brain. “Parenteral”includes subcutaneous, intradermal, intramuscular, intravenous,intra-arterial, intrathecal, and other injection or infusion techniques,without limitation.

Suitable choices in amounts and timing of doses, formulation, and routesof administration can be made with the goals of achieving a favorableresponse in the subject (i.e., efficacy or therapeutic), and avoidingundue toxicity or other harm thereto (i.e., safety). Administration maybe by bolus or by continuous infusion. Bolus refers to administration ofa drug (e.g., by injection) in a defined quantity (called a bolus) overa period of time. Continuous infusion refers to continuing substantiallyuninterrupted the introduction of a solution into a blood vessel for aspecified period of time. A bolus of the formulation administered onlyonce to a subject is a convenient dosing schedule, although in the caseof achieving an effective concentration of the cytoprotective compoundin the brain more frequent administration may be required. Treatment mayinvolve a continuous infusion (e.g., for 3 hr after stroke) or a slowinfusion (e.g., for 24 hr to 72 hr when given within 6 hr of stroke).Alternatively, it may be administered every other day, once a week, oronce a month. Dosage levels of active ingredients in a pharmaceuticalcomposition can also be varied so as to achieve a transient or sustainedconcentration of the compound or derivative thereof in a subject and toresult in the desired therapeutic response.

The pharmaceutical compositions of the invention may be administered asa formulation, which is adapted for direct application to the centralnervous system, or suitable for passage through the gut or bloodcirculation. Alternatively, pharmaceutical compositions may be added tothe culture medium. In addition to active compound, such compositionsmay contain pharmaceutically acceptable carriers and other ingredientsknown to facilitate administration and/or enhance uptake. It may beadministered in a single dose or in multiple doses, which areadministered at different times. A unit dose of the composition is anamount of the cytoprotective compounds which provides effectivecytoprotection, inhibits apoptosis or cell death, and/or promotes cellsurvival. Measurement of such values can be performed with standardtechniques well known in the art, e.g., clinical laboratories routinelydetermine these values with standard assays and hematologists classifythem as normal or abnormal depending on the situation.

When administered to a subject in vivo, the pharmaceutical compositionstypically contain a therapeutically effective amount of the activecytoprotective compound. A therapeutically effective amount is the totalamount of the cytoprotective compound that achieves the desiredcytoprotective effect. Depending on the species of the subject ordisease to be treated, the therapeutic amount may be about 0.01 mg/kg/hrto about 1.1 mg/kg/hr if administered by continuous infusion over 4 hourto 96 hour, to as little as about 0.01 mg/kg/hr to about 0.10 mg/kg/hrfor about 24 hours. Preferably, the therapeutic dose would beadministered by continuous infusion for about 4 to about 72 hours. Morepreferably, by continuous infusion for about 4 to about 48 hours. Morepreferably, by continuous infusion for about 12 to about 48 hours. Morepreferably, by continuous infusion for about 12 to about 36 hours. Morepreferably, by continuous infusion for about 4 to about 36 hours. Morepreferably, by continuous infusion for about 12 to about 24 hours. Mostpreferably, by continuous infusion for about 24 hours. In otherexamples, a therapeutically effective amount for bolus administrationcan typically be 2 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less,0.04 mg/kg or less, 0.03 mg/kg or less, 0.02 mg/kg or less, 0.01 mg/kgor less, or 0.005 mg/kg or less. Typically, the therapeutic amount maybe based on titering to a blood level amount of the cytoprotectivecompound of about 0.01 μg/ml to about 1.6 μg/ml, preferably from about0.01 μg/ml to about 0.5 μg/ml. It is also within the skill of the art tostart doses at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. It is likewise within the skill of the artto determine optimal concentrations of variants to achieve the desiredeffects in the in vitro and ex vivo preparations of the invention.Depending on initial assay results, optimal concentrations can be in therange of, e.g., about 1-1,000 nM or about 1-200 μM depending on thegeneral nature of the compound.

EXAMPLES

The following examples are provided to further illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Example 1: Activated Protein C Cleaves a Synthetic PAR1 Peptide(TR33-62) at Arg⁴⁶

Peptide name Peptide sequence TFLL TFLLRNPNDK (TR42-51 with S42T)(SEQ ID NO: 8) NPND (TR47-66) NPNDKYEPFWEDEEKNESGL (SEQ ID NO: 4)scr-NPND GDENENEKPNWYELKEPDSF (scrambled TR47-66) (SEQ ID NO: 9) TR24-41TRARRPESKATNATLDPR (SEQ ID NO: 10) TR24-46 TRARRPESKATNATLDPRSFLLR(SEQ ID NO: 11) TR42-66 SFLLRNPNDKYEPFWEDEEKNESGL (SEQ ID NO: 12)TR33-62 ATNATLDPRSFLLRNPNDKYEPFWEDEEKN (SEQ ID NO: 13)

To characterize APC cleavage sites in PAR1 N-terminal tail, we studiedthe HPLC patterns from cleavage of a synthetic peptide (“TR33-62”)comprising residues 33-62 of the PAR1 N-terminus by thrombin and APC andgot the mass spec values for each observed peak. The results are shownin FIG. 2. In the HPLC profiles, the uncleaved substrate TR33-62 (peakE) gave the correct mass of 3,651 and the two fragments produced bythrombin (peaks A and D) corresponded to masses of 1,001 and 2,670 forcleavage at Arg⁴¹ as expected. In contrast to thrombin, APC gave fourcleavage peaks (A and D, plus B and C) which corresponded to masses forcleavages at both Arg⁴¹ (peaks A and D) and at Arg46 (peaks B and C).Thus, APC cleaves this PAR1 peptide at Arg⁴⁶ as well as at Arg⁴¹.

Methods: Cleavage by APC or thrombin of a synthetic (“TR33-62”) PAR1peptide (Biosynthesis) comprising residues 33-62 of the PAR1 N-terminus(SEQ ID NO:13) was analyzed by reverse phase HPLC on a C18 column withabsorbance monitoring at 214 nm. APC (500 nM) or thrombin (5 nM) wereincubated with TR33-62 (100 in Hepes (20 mM) buffered saline (147 mMNaCl/4 mM KCl) pH 7.4 (HBS) for various times after which 20 μl of 1.2 Mperchloric acid was added to stop the reaction. Samples were subjectedto reverse phase HPLC on a C18 column while the eluent was monitored at214 nm during a 0%-67% gradient of acetonitrile in water containing 0.1%trifluoroacetic acid. Peptide fragments were collected and subjected toMALDI-TOF (Scripps mass spectrometry core facility) analysis.

Example 2: Activated Protein C (APC) Cleaves PAR1 at Arg⁴⁶ onTransfected HEK-293 Cells

To facilitate analysis of APC-mediated PAR-1 activation, we created afull-length PAR-1 construct linked at the N-terminus to an alkalinephosphatase reporter group (SEAP). Selection of stable cell linesexpressing both endothelial protein C receptor (EPCR) (wt or E86A) andSEAP-PAR1 allowed for a reliable and reproducible analysis ofEPCR-dependent PAR-1 activation by APC. The results are shown in FIG. 3.Mass-spec analysis of cleavage fragments generated by APC of a syntheticPAR-1 N-terminal tail peptide (TR33-62) identified both Arg⁴¹ and Arg⁴⁶as potential cleavage sites for APC proteolysis, as noted above. Tocharacterize the cleavage of PAR1 by APC on cells, SEAP-PAR1 mutantswere generated with either the thrombin cleavage site Arg⁴¹ mutated(R4Q-SEAP-PAR1) or the APC cleavage site Arg⁴⁶ mutated(R46AQ-SEAP-PAR1). R41Q-SEAP-PAR1 was cleaved by APC in the presence ofEPCR. This surprising result was also obtained for R41A-SEAP-PAR1.Mutagenesis of R46Q in SEAP-PAR1 did not eliminate PAR1 cleavage by APCwhereas mutagenesis of both R41Q/R46Q in SEAP-PAR1 eliminated PAR-1cleavage by APC. Interestingly, APC did not cleave R41A-SEAP-PAR1 in theabsence of EPCR whereas it did cleave wt-SEAP-PAR1 and R46Q-SEAP-PAR1,suggesting that in the absence of EPCR, APC can still cleave PAR-1 atArg⁴¹ but not at Arg⁴⁶.

These data identified PAR-1 residue Arg⁴⁶ as an alternative to Arg⁴¹ forproteolysis of intact PAR1 on cell surfaces by APC. Thrombin did notshow any cleavage of R41Q-PAR1, i.e., it did not cleave PAR1 on cells atArg⁴⁶. The relatively nonspecific protease, elastase, cleaved all PAR1constructs on cells with similar efficacy, independent of EPCR. Asimilar set of PAR1 constructs where Ala replaced Arg⁴¹ and/or Arg⁴⁶showed essentially identical results. We conclude that EPCR-dependentPAR-1 cleavage at Arg⁴⁶ by APC could result in cytoprotective signalingwhereas PAR-1 cleavage at Arg⁴¹ by thrombin results in proinflammatoryPAR-1 signaling.

Methods: A PAR-1 cleavage reporter construct was made in which asecreted alkaline phosphatase (SEAP) was fused to the N-terminus ofPAR-1 which is released by proteolysis by APC or thrombin at residueArg⁴¹ or Arg⁴⁶ as described in Mosnier et al., Blood. 2009;113:5970-5978. SEAP-PAR1, either in the presence or absence of wt-EPCR,were transfected into HEK-293 cells to obtain stable cells expressingSEAP-PAR1 or wt-EPCR/SEAP-PAR1. Mutations in SEAP-PAR1 were introducedusing the Quickchange method (Agilent). Cells were grown in 96-wellsplates to confluency in Dulbeco's modified Eagle media (DMEM): Ham'sF-12 (Invitrogen) supplemented with penicillin-streptomycin-glutamine(PSG) (Gibco) and 10% fetal bovine serum (Omega) in a humidifiedatmosphere at 37° C. and 5% CO₂. Cells were washed with Hank's balancedsalt solution (HBSS; Invitrogen) supplemented with 1.3 mM CaCl₂, 0.6 mMMgCl₂ and 0.1% endotoxin free BSA (Calbiochem) and incubated with APC.After 60 min, SEAP release was determined using 1-step PNPP (Pierce).Values were corrected for background activity derived from the samecells incubated in the absence of APC and expressed as a percentage oftotal SEAP available on the cell membrane.

Example 3: Activated Protein C Cleaves PAR1 at Arg⁴⁶ on EA.hy.926Endothelial Cells

We showed above that Activated Protein C (APC) cleaves a synthetic PAR1N-terminal tail peptide at Are and Arg⁴⁶. On HEK293 cells APC cleaved aSEAP-PAR1 fusion construct at Arg⁴¹ and Arg⁴⁶ when cells wereco-transfected with wt-EPCR. To determine whether APC cleaves endogenousPAR1 at the non-canonical Arg⁴⁶ site on endothelial cells in thepresence of endogenous endothelial protein C receptor (EPCR), anti-PAR1antibodies with defined epitopes were used to characterize the presenceor disappearance of specific PAR1 epitopes on the cell surface ofEA.hy.926 endothelial cells upon incubation with thrombin or APC.Specifically, PAR1 peptides (Biosynthesis) were synthesized and purifiedto >95%. Peptides were coated to 96-well maxisorp plates (Nunc) at 10 μMin carbonate buffer pH 9.0. Plates were blocked with Tris (50 mM)buffered saline (150 mM NaCl) pH 7.4 (TBS) containing 3% BSA.Subsequently, antibodies were incubated at 2 μg/ml in blocking buffer.After washing with TBS/0.1% Tween appropriate HRP-labeled secondaryantibodies (DAKO) were added at 1:1000 dilution in blocking buffer.Plates were then washed with TBS/0.1% Tween and developed witho-phenylenediamine dihydrochloride (OPD) for the appropriate time,reactions were stopped by addition of ⅓ volume 1M H₂SO₄ and absorbancewas determined at 490 nm.

The results obtained from the study are shown in FIG. 4. First, theepitopes of various PAR1 antibodies were validated using solid-phasebinding assay with immobilized PAR1 peptides. Goat anti-PAR1 antibodies(Ab87 and Origene), raised against the PAR1 peptide residues 1-100 andresidues 24-37 respectively, reacted only with PAR1 peptides TR24-41 andTR24-46 but not with PAR1 peptides TR42-66 and TR47-66. Also, these goatanti-PAR1 antibodies did not react with PAR1 peptide 33-62 indicatingthat the epitope of these antibodies requires the PAR1 sequence 24-32which must represent most or all of the epitope for these antibodies.Thus, these antibodies are cleavage sensitive antibodies and theirsignals on cells are anticipated to diminish when APC or thrombincleaves PAR1 to liberate soluble activation peptides (e.g., residues24-41, 24-46, and 42-46) regardless of whether that cleavage occurs atArg⁴¹ or at Arg⁴⁶. Monoclonal anti-PAR1 antibody ATAP2 has an epitopeconfined within residues 41-55 of PAR1 (Brass et al., J Biol Chem. 1994;269:2943-2952).

In a solid-phase binding assay with immobilized PAR1 peptides ATAP2 didnot react with the N-terminal cleavage products TR24-41 or TR24-46. Incontrast, ATAP2 did bind to PAR1 peptide TR42-66 but not to the shorterpeptide TR47-66 indicating that critical for epitope recognition areresidues within the PAR1 sequence 42-46. Thus, ATAP2 is a cleavage sitesensitive antibody. Its signal on cells is anticipated to diminish whenAPC cleaves PAR1 at Arg⁴⁶ but not when PAR1 is cleaved at Arg⁴¹,therefore in the absence of a signal for the goat anti-PAR1 antibodies(ab87 or Origene) which reports cleavage of the PAR1 N-terminal tail,the presence or absence of the ATAP2 signal on cells will report whetherPAR1 is cleaved at Arg⁴¹ or at Arg⁴⁶, respectively. Monoclonal anti-PAR1antibody WEDE15 has an epitope confined within residues 51-64 of PAR1(Brass et al., J. Biol. Chem. 269:2943-2952, 1994). In a solid-phasebinding assay with immobilized PAR1 peptides WEDE15 did not react withthe N-terminal cleavage products TR24-41 or TR24-46. WEDE15 did bind toPAR1 peptides TR42-66, TR47-66 and TR33-62 confirming that the criticalepitope residues reside within the PAR1 sequence 47-62. Thus, WEDE15binding to PAR1 is insensitive to cleavage at Arg⁴⁶ by APC. The WEDE15binding signal on cells is anticipated to remain unchanged upon APC orthrombin cleavage of PAR1, regardless of whether that cleavage occurs atArg⁴¹ or at Arg⁴⁶. Therefore, WEDE15 will report the total of availablePAR1 molecules on the surface of the cell membrane and can be used fornormalization of the signals for the goat anti-PAR1 antibodies (ab87 orOrigene) and ATAP2 whose epitopes are sensitive to proteolysis of PAR1.

Subsequently, these antibodies were used in an on cell Western (OCW)immunoassay on EA.hy.926 endothelial cells to determine whether APCcleaves PAR1 at Arg⁴⁶ in the presence of endogenous PAR1 and EPCRexpression. In this study, confluent EA.hy.926 cells plated in blackclear bottom 96-well tissue culture treated plates were incubated for 1hr with 0.5 nM thrombin or 25 nM APC. Incubations were performed at 4°C. to minimize receptor recycling and internalization. After fixationand staining, antibody signals were normalized for total cell numberusing the cell permeable nuclear dye Draq5 and antibody signals for thegoat anti-PAR1 antibodies (Ab87 and Origene) and ATAP2 were expressed asa fraction of the signal obtained for WEDE15 reflecting the total PAR1present on the cell surface.

Results obtained from this study are shown in FIG. 5. Incubation of theEA.hy.926 endothelial cells with thrombin or APC resulted in 60-75%reduction of the cleavage sensitive goat anti-PAR1 antibody (Ab87 andOrigene) signals indicating that both thrombin and APC cleaved PAR1. Thesignal for ATAP2 was not significantly different for that of WEDE15 uponincubation of the cells with thrombin indicating that thrombin hadcleaved PAR1 at the canonical Arg⁴¹ cleavage site. In contrast, theATAP2 signal decreased 50% compared to WEDE15 upon incubation of thecells with APC indicating that significant APC-mediated cleavage of PAR1at the non-canonical cleavage site at Arg⁴⁶ had occurred.

Thus, on untransfected EA.hy.926 endothelial cells expressing endogenousEPCR and PAR1, APC cleaves PAR1 at Arg⁴⁶ whereas thrombin cleaves PAR1at Arg⁴¹ giving rise to different new N-termini starting at Ser⁴² afterthrombin cleavage or starting at Asn⁴⁷ after APC cleavage. Because thenew N-terminus acts as a tethered ligand (agonist) for activation of thePAR1 receptor and induction of signaling pathways, this difference innew N-terminal residues, i.e., SFLLRN (SEQ ID NO:21)-etc. for thrombinversus NPNDKY (SEQ ID NO:14)-etc. for APC, shows that thrombin and APCcreate different tethered-ligands (agonists). Different receptoragonists are known to be able to activate G protein-coupled receptors(GPCR) differently, resulting in the induction of different cellsignaling pathways. It was therefore conceivable that, because APC andthrombin create different PAR1 agonists, APC and thrombin activate PAR1differently to such an extent that different cell signaling pathwayscould be activated, depending on whether the PAR1 agonist is derivedfrom a N-terminus that starts with SEQ ID NO:21 or one that starts withSEQ ID NO:14.

Detailed methods and steps used for the study shown in FIG. 5 are asfollows: EA.hy.926 cells (ATCC: CRL-2922) were grown in black clearbottom 96 well plates (Nunc) in DMEM (Invitrogen #12430) supplementedwith glutamax (Invitrogen #35050) and 10% FBS (Hyclone #35-011-CV) in a37° C. cell incubator with a humidified atmosphere and 5% CO₂ in airuntil confluency. Cells were prechilled to 4° C. and treated for 1 hrwith thrombin (0.5 nM) or APC (25 nM) in Hanks buffered salt solution(HBSS; Invitrogen) supplemented with 0.1% endotoxin free BSA(Calbiochem) 1.3 mM CaCl₂ and 0.6 mM MgCl₂. Subsequently cells werefixed with methanol free 4% para-formaldehyde (Pierce), washed withphosphate buffered saline (PBS; Invitrogen) and blocked with Odysseyblocking buffer (Licor). Cells were incubated with 2.5 μg/ml goatanti-PAR1 antibodies ab86 (Abeam #ab66068) or Origene (Origene#TA305911) or with 10 μg/ml ATAP2 or WEDE15 in Odyssey blocking buffer.After washing with PBS/0.1% Tween ( 1/600) IRDye 800CW-labelledsecondary goat anti-mouse or donkey anti-goat antibodies (Licor) wereadded to the cells in Odyssey blocking buffer and 1/10,000 Draq5(Biostatus) for cell normalization. After final washing with PBS/0.1%Tween, IR fluorescence of bound antibodies was determined using theOdyssey (Licor). Background fluorescence was determined by omitting theprimary antibody and subtracting background values from all observedvalues.

Example 4: Activation of Cell Signaling (ERK 1/2) by APC, Thrombin andTR42-51 (TRAP; TFLL) and TR47-66 (NPND) Peptides on EA.hy.926Endothelial Cells

To assess whether PAR1 cleaved at Arg⁴⁶ would generate a PAR1 N-terminusthat is capable of promoting PAR1 signaling, we studied the effects ofsynthetic peptides on ERK1/2 phosphorylation in EAhy926 endothelialcells. ERK1/2 phosphorylation is an established marker of PAR1-dependentsignaling by thrombin and APC on endothelial cells (Riewald et al.,Science. 2002; 296:1880-1882). The peptides included a Thrombin ReceptorActivating Peptide (“TRAP”; aka “TFLL”) reflecting the sequence of PAR1residues 42-51 (SEQ ID NO:8), a peptide comprising residues 47-66(TR47-66) that would be generated by APC cleavage at Arg⁴⁶ (SEQ IDNO:4), and a control scrambled TR47-66 peptide (designated“scr-TR47-66”) containing scrambled residues 47-66 (SEQ ID NO:9).Relative phosphorylation of ERK1/2 was determined using the LI-COROdyssey infrared imaging system which permits quantitative analysis overa wide dynamic range.

As shown in FIG. 6, the Li-COR Odyssey permits simultaneous detection ofmultiple signals when detecting antibodies yield either red or greensignals; notably each color for different antibodies for overlappingbands can be deconvoluted from the total signal. In this figure (left),anti-pT202Y204-ERK1/2 antibodies appear green while total anti-ERK1/2antibodies appear red, and orange intensity reflects overlaps. Cellswere treated for 5 min with 10 nM APC or thrombin or 10 μM TFLL peptideor 50 μM TR47-66 or scr-TR47-66 prior to cell lysis and immunoblottinganalysis. The signal for phosphorylated ERK1/2 (green channel) wasnormalized to the signal for total ERK1/2 antigen (red channel) toquantify relative phosphorylation as a ratio, and the results are seenin the bar graph. Data in the bar graph show that thrombin and TRAPcaused a 3-fold increase in ERK1/2 phosphorylation and that APC andTR47-66 caused a 1.5-fold increase in phosphorylation. The controlscr-TR47-66 peptide (scrambled residues 47-66) caused no increase. Thus,these data suggest that the amino acid sequence of PAR1 that has beencleaved at Arg⁴⁶ can cause signaling, i.e. ERK1/2 phosphorylation, thatis typical of PAR1-dependent signaling initiated by APC or thrombin.These data indicate that APC can initiate signaling by cleavage at Arg⁴⁶in PAR1.

Methods: EA.hy.926 cells (ATCC: CRL-2922) were grown in 6-well plates inDMEM (Invitrogen #12430) supplemented with glutamax (Invitrogen #35050)and 10% FBS (Hyclone #35-011-CV) in a 37° C. cell incubator with ahumidified atmosphere and 5% CO₂ in air until confluency. Cells wereserum starved overnight and treated for 5 min with PAR1 peptides TFLL(10 μM), NPND (250 μM) or scr-NPND (250 μM) or with vehicle controlthrombin or APC in thrombin (10 nM) or APC (10 nM) in serum free media.Subsequently cells were resuspended in lysis buffer with protein andphosphatase inhibitors, cell lysates were applied to SDS-PAGE gelelectrophoresis on 10% Bis-Tris gels with MOPS running buffer(Invitrogen) and transferred to PVDF membrane (Millipore) for Westernblot analysis. Blots were blocked in Odyssey blocking buffer (Licor) andincubated with a combination of mouse anti-ERK1/2 ( 1/2000; CellSignaling #3A7) and rabbit anti-pT202/204 ERK1/2 ( 1/1000; cellsignaling #D13.14.4E) antibodies in Odyssey blocking buffer (Licor).After washing, blots were incubated with IRDye680-labeled donkeyanti-mouse ( 1/10,000; Licor) and IRDye800CW-labeled donkey anti-rabbit( 1/10,000), washed again with PBS/0.1% Tween and IR fluorescence ofbound antibodies was determined using the Odyssey (Licor).

Example 5: Preferential Activation of Akt by the TR47-66 Peptide VersusPreferential Activation of ERK1/2 by the TR42-51 Peptide (TFLL) onEA.hy.926 Endothelial Cells

Activated Protein C (APC) cleaves a synthetic PAR1 tail peptide at Arg⁴¹and Arg⁴⁶; furthermore, APC cleaves SEAP-PAR1 mutants with the mutationsR41A or R41Q but not SEAP-PAR1 with both R41Q/R46Q mutations. Thrombindoes not cleave PAR1 at Arg⁴⁶. It was thus hypothesized that a new PAR1N-terminus beginning at residue Asn-47 promotes PAR1 signaling and APCcytoprotective activities. Since phosphorylation of signaling networkcomponents is useful for monitoring activation of signaling pathways,the effects of synthetic PAR1 peptides on Akt and ERK1/2 phosphorylationin EA.hy.926 human endothelial cells were studied. Cells were treatedfor 0, 5 or 30 min with peptides prior to cell fixation,permeabilization of cells and subsequent immunoblotting analysis(“in-cell Western blotting”). Relative phosphorylation of Akt at Ser⁴⁷³(pSer⁴⁷³-Akt) or of ERK1/2 at Thr202/204 (pERK1/2) in endothelial cellswas quantified using the LI-COR Odyssey infrared (IR) fluorescenceimaging system which permits “in cell” Western blots. The Odysseypermits simultaneous quantification of different antibodies that yieldeither red or green signals.

Results obtained from the study are shown in FIG. 7. As seen from thefigure (panel A), a cell marker (red intensity) can be used to quantifyrelative numbers of cells in each well, permitting a very accuratenormalized ratio calculation of the relative phosphorylation of Akt(green intensity for pSer⁴⁷³-Akt in panel A) or similarly of ERK1/2(data not shown). The peptides studied included: 1) “TRAP” (ThrombinReceptor Activating Peptide) (“TFLL”) that represents PAR1 residues(42-TFLLRNPNDK-51, with Tin place of S42; see Vu et al., Cell. 1991;64:1057-1068) (SEQ ID NO:8); 2) “NPND” designating PAR1 residues47-NPNDKYEPFWEDEEKNESGL-66 (SEQ ID NO:4) comparable to the newN-terminal sequence appearing after cleavage of PAR1 at Arg⁴⁶ by APC;and 3) “scr-NPND”, a negative control for NPND containing scrambled PAR1residues 47-66 (SEQ ID NO:9). The NPND peptide, comprising PAR1 residues47-66, caused a time-dependent sustained increase in pSer⁴⁷³-Aktcompared to TFLL and scr-NPND peptides which had essentially no effecton Akt phosphorylation (panel B). In contrast, the TRAP compound, TFLLcomprising PAR1 residues 42-51, quickly but transiently stimulatedERK1/2 phosphorylation, compared to NPND and scr-NPND which hadessentially no effect on ERK1/2 phosphorylation (panel C). These dataindicate that cleavage at Arg⁴¹ by thrombin or APC generates a newN-terminus which is a classical TRAP whereas APC's cleavage at Arg⁴⁶generates a new N-terminus beginning with NPND-which can initiate novelsignaling that causes phosphorylation of Akt, but not of ERK1/2.Activation of the Akt-survival pathway is known to providecytoprotective activities that enable cells to survive potentiallylethal stimuli (e.g., hypoxia, radiation, oxidative stress, etc.).

Methods: EA.hy.926 cells (ATCC: CRL-2922) were grown in black clearbottom 96 well plates (Nunc) in DMEM (Invitrogen #12430) supplementedwith glutamax (Invitrogen #35050) and 10% FBS (Hyclone #35-011-CV) in a37° C. cell incubator with a humidified atmosphere and 5% CO₂ in airuntil confluency. Cells were treated for 0, 5 or 30 min with PAR1peptides TFLL (50 μM), NPND (250 μM) or scr-NPND (250 μM) or withvehicle in serum free media. Subsequently cells were fixed with methanolfree 4% para-formaldehyde (Pierce), washed with phosphate bufferedsaline (PBS; Invitrogen) containing 1% triton X-100 and blocked withOdyssey blocking buffer (Licor). Cells were incubated with rabbitanti-pT202/Y204 ERK1/2 ( 1/1000; Cell signaling D13.14.4E) or rabbitanti-pSer⁴⁷³ ( 1/100; cell signaling D9E) with Odyssey blocking buffer.After washing with PBS/0.1% Tween ( 1/800), IRDye 800CW-labelledsecondary goat anti-rabbit antibodies (Licor) were added to the cells inOdyssey blocking buffer and 1/10,000 Draq5 (Biostatus) for cellnormalization. After final washing with PBS/0.1% Tween IR, thefluorescence of bound antibodies was determined using the Odyssey(Licor). Background fluorescence was determined by omitting the primaryantibody and subtracting values for controls from all other observedvalues.

Example 6: TR47-66 Peptide Conveys Anti-Apoptotic Effects on EA.hy.926Endothelial Cells

Activated Protein C (APC) conveys anti-apoptotic activity to EA.hy.926cells that requires functional PAR1 receptor and APC binding to theendothelial protein C receptor (EPCR). See, e.g., Mosnier et al.,Biochem J. 2003; 373:65-70; and Mosnier et al., Blood. 2007;109:3161-3172. To determine whether APC's anti-apoptotic activity is theresult of PAR1 cleavage at Arg⁴¹ or at Arg⁴⁶, PAR1 peptides representingthe newly generated N-termini after cleavage at Arg⁴¹ (TFLL) or at Arg⁴⁶(NPND) were evaluated for their ability to inhibit staurosporine-inducedendothelial cell apoptosis. It was found that the NPND peptidesignificantly inhibited endothelial cell apoptosis (FIG. 8). Thisdemonstrates that non-canonical cleavage of PAR1 at Arg⁴⁶ generates anew, signaling competent N-terminus that conveys anti-apoptoticactivity. In contrast, the TFLL peptide representing the new N-terminusof PAR1 resulting from canonical cleavage of PAR1 at Arg⁴¹ and thecontrol scrambled sequence scr-NPND peptide did not convey detectableanti-apoptotic effects (FIG. 8).

To further confirm activation of Akt by the TR47-66 peptide,Akt-mediated inactivation of glycogen synthase kinase 3β (GSK3β) viaphosphorylation at Ser9 was determined. GSK3β is a well-known downstreamsubstrate for Akt. It was found that TR47-66 induced significantSer9-GSK3β phosphorylation starting at 30 min with a time-course thatfell well within the time course of TR47-mediated Akt activation.Similar to phosphorylation of Akt at Ser473, no phosphorylation of GSK3βat Ser9 was observed for cells treated with the scrambled controlpeptide. This indicates that the effects of the TR47-66 peptide werespecific for the newly created N-tethered ligand sequence of PAR1 aftercleavage at Arg46.

Additionally, it was determined that activation of Akt by TR47-66 wasdependent on PAR1 since the peptide failed to induce Akt phosphorylationat Ser473 in the presence of the PAR1 inhibitor SCH79797. Similarly,induction of Ser9-GSK3β phosphorylation by TR47-66 required PAR1 sincethis effect was inhibited by the PAR1 antagonist SCH79797. Thus, apeptide with the sequence of the new N-terminus of PAR1 generated uponcleavage at Arg46 induced PAR1-dependent activation of Akt andPAR1-dependent phosphorylation of GSK3β, strongly suggesting thatactivation of PAR1 at Arg46 creates a novel functional tethered ligandthat is capable of PAR1-dependent activation of specific cell signalingpathways.

Methods: EA.hy.926 cells (ATCC: CRL-2922) were grown in 24 well platesin DMEM (Invitrogen #12430) supplemented with glutamax (Invitrogen#35050) and 10% FBS (Hyclone #35-011-CV) in a 37° C. cell incubator witha humidified atmosphere and 5% CO₂ in air until confluency. Cells weretreated for 4 hrs with vehicle control, 50 μM NPND, 50 μM scr-NPND or 10μM TFLL after which staurosporine (10 μM) and the Apopercentage dye(Biocolor) were added according the manufacturer's instructions. After 1hr cells were washed with 2×1 ml PBS and 110 μl of the dye release agentwas added to the cells. Subsequently 100 μl aliquots were transferred toa black 96 well plate and fluorescence was measured (530 nmexcitation/580 nm emission). Apoptosis was expressed as a percentage ofcells treated with 10 mM H₂O₂ for which apoptosis was set at 100%.

Example 7: TR47-66 Peptide Mimics Signaling Induced by APC, not that byThrombin

Further studies were performed to confirm that the TR47-66 peptidemimics APC-induced signaling but does not mimic thrombin-induced orTRAP-induced cell signaling, as indicated in the Examples above.Classical activation of PAR1 by thrombin or TRAP results in theactivation of the mitogen-activated protein kinase (MAPK) pathway astypically demonstrated by rapid phosphorylation ofextracellular-signal-regulated kinases 1 and 2 (ERK1/2) at Thr202 andTyr204 or Thr185 and Tyr187, respectively. We observed that, consistentwith earlier reports, APC induced a transient but modest activation ofERK1/2. Interestingly, TR47-66 failed to induce any noticeableactivation of ERK1/2 under conditions where TRAP and APC did so. Thus,TR47-66 does not activate the classical PAR1 MAPK pathway that isactivated by thrombin and TRAP. Remarkably, TR47-66 induced activationof Akt with a time course that mimicked APC's robust activation of Akt.In contrast and reflecting the striking functional selectivity ofTR47-66 and APC, neither thrombin nor TRAP induced Ser473-Aktphosphorylation under the employed experimental conditions. Similar toactivation of Akt, APC induced a sustained phosphorylation of GSK3β atSer9, mimicking the effect of TR47-66. Unlike this effect of APC andTR47-66, incubation of endothelial cells with TRAP did not result inSer9-GSK3β phosphorylation. Together these signaling data indicated thatthe N-terminus generated by cleavage of PAR1 at Arg41 causes activationof the MAPK pathway whereas the N-terminus generated by cleavage of PAR1at Arg46 causes activation of the Akt pathway. Thus, the differentN-termini that arise from different cleavages of PAR1 by APC are biasedagonists with remarkable functional selectivity.

Example 8: TR47-66 Induced Vascular-Endothelial Protective Effects InVitro and In Vivo

Employing β-arrestin-mediated signaling versus G protein-dependentsignaling is a hallmark of biased signaling by GPCRs. Recently, PAR1 wasshown to exhibit biased signaling because activation of Rac1 by APC andAPC-mediated endothelial barrier protection requires β-arrestin-2 anddishevelled-2 scaffolding whereas thrombin-induced vascular leakage andRhoA activation requires G proteins but not β-arrestins (Soh et al.,Proc. Natl. Acad. Sci. U.S.A 2011; 108:E1372-E1380). Consistently, weobserved that the TR47-66 peptide but not the scrTR47 control peptideinduced APC-like activation of Rac1. We also observed that, mimickingthe well known vasculoprotective activity of APC, the TR47-66 peptideprotected confluent endothelial barriers against thrombin-inducedpermeability, whereas the control scrTR47 peptide was without effect.

To probe whether TR47-66 could induce vascular protective effects invivo, a modification of the modified Miles assay was used in whichvascular permeability is measured by the extravasation of Evans blue dyein the skin induced by local subcutaneous injection of vascularendothelial growth factor (VEGF)165. Immunocompetent SKH1 hairless micewere used to avoid the need for hair removal that often can result inartifactual leakage due to inflammation of the skin. Evans blueextravasation in the skin was quantified using the Odyssey near-infraredfluorescent imager at 700 nm. TR47-66 peptide or the control scrTR47peptide were injected i.v. in the retro-orbital sinus 5 min before localsubcutaneous injection of recombinant murine VEGF or BSA control and 30min after i.v. administration of Evans blue. In the absence of TR47-66(PBS control), VEGF induced clearly distinguishable areas of Evans blueextravasation, whereas after injection of BSA vehicle control only theneedle points marking the injection site could be observed.Quantification of Evans blue extravasation by near infrared fluorescenceat 700 nm eliminated the time consuming need for Evans blue extractionfrom punch biopsy and provided reliable and reproducible results. TheTR47-66 peptide significantly decreased vascular leakage by 45% comparedto PBS control, whereas vascular leakage in the presence of the scrTR47control peptide was indiscriminate from PBS control. Neither TR47-66 norscrTR47 affected vascular leakage in the absence of VEGF. Thus, likeAPC, the TR47 peptide causes activation of Rac1, stabilizes endothelialbarriers in vitro, and in vivo can markedly reduce vascular leakage.

Some assays employed in this study were performed as follow. Radactivation. The pGEXTK-PAK1 70-117 construct encoding a GST fusion top21-activated kinase (PAK-1)-binding domain (PBD) was kindly madeavailable by Dr. J Chernoff (Addgene plasmid 12217). GST-PAK1 waspurified from transformed BL21 (DE3) Escherichia coli using B-Per lysisbuffer (Pierce) with lysozyme, DNAse I and Halt EDTA-free proteaseinhibitor cocktail (Pierce) on Glutathione-agarose according to themanufacturer's recommendation. Pull down of active Rac1 was performed asdescribed in Pellegrin et al., Curr. Protoc. Cell Biol. 2008; Chapter14: Unit 14.8. Briefly, endothelial EA.hy.926 cells (5*10⁶ per plate)were grown in 100-mm dishes for 48 hr and serum starved overnight beforeaddition of peptides (50 μM) for 30 or 180 min. Lysates (2 mg) weremixed with GST-PAK1 Glutathione-agarose (150 μg) and after washingactive GTP-Rac1 was eluted from GST-PAK1 Glutathione-agarose by boilingin reducing SDS sample buffer (LI-COR). Active GTP-Rac1 was resolved on12% SDS PAGE, transferred to Immobilon-FL PVDF membrane, andimmunoblotted with a mouse anti-Rac1 antibody (BD Bioscience) and IRDye800CW donkey anti-mouse secondary antibodies (LI-COR). Immunoblots werescanned on the Odyssey Imager (LI-COR). Quantification of integratedfluorescence intensity (K counts) was done using Odyssey ApplicationSoftware v3.0 (LI-COR).

Endothelial Barrier Protection.

Permeability of endothelial cell barrier function was determined asdescribed with minor modifications. Briefly, EA.hy.926 endothelial cells(5*10⁴ cells/well) were grown on polycarbonate membrane Transwellinserts (Costar, 3 μm pore size, 12 mm diameter). When confluent, cellswere incubated with APC (20 nM), TR47 (50 μM), or scrTR47 (50 μM) for 4hr in serum-free media with 0.1% BSA (fatty acid poor and endotoxin freefraction V, Calbiochem). After incubation with thrombin (10 nM) for 10min, the media in the inner chamber was replaced with complete mediumcontaining 4% BSA and 0.67 mg/ml Evans blue. Endothelial cellpermeability was determined by absorbance of Evans blue in the outerchamber at 650 nm. Permeability was expressed as the fold change inabsorbance compared to that in the absence of thrombin (normalized to1).

In Vivo Vascular Permeability Assay.

The study was approved by the Institutional Animal Care and UseCommittee of The Scripps Research Institute and complied with NationalInstitutes of Health guidelines. SKIH1-E hairless male (6 to 8 weeksold) were from Charles River Labs (Wilmington, Mass.). Vascularpermeability was determined using a known VEGF-induced leakage model(Sanna et al., Nat. Chem. Biol. 2006; 2(8):434-441; and Miles et al., JPhysiol 1952; 118(2):228-257) with some modifications. Briefly, 1004 ofa sterile-filtered solution containing 0.5% (w/v) Evans blue (Sigma) in0.9% NaCl (Sigma) was injected in isofluran-anesthetized mice. After 30min, 50 μL of peptides (125 μg; TR47 or scrTR47) or PBS were injectedintravenously in the retro-orbital sinus of mice anesthetized withKetamin-Xylazin (100 and 10 mg/kg, respectively). After 5 min, micereceived subcutaneously 15 mL of 75 ng/injection recombinant mouseVEGF165 (BioVision, Milpitas, Calif.) in 0.1% BSA-PBS (3 sites on theright side of the abdomen) or vehicle (2 sites on the left side). After30 min, mice were sacrificed, photographed, and Evans blue extravasationin the skin was determined using the Odyssey Imager (LI-COR Biosciences,Lincoln, Nebr.) in the 700 nm channel with 4 mm offset. Quantificationof the intensity of the Evans blue dye signal was done using the OdysseyApplication Software version 3.0. A mean value for 3 data points (VEGF)or 2 data points (BSA) (injection sites) was made for each mouse andnormalized to the VEGF sites in PBS-injected mice. In total, 5independent experiments were performed using a total of 23 mice (n=11for PBS, n=6 for TR47 and n=6 for scrTR47).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All publications, databases, GenBank sequences, patents, and patentapplications cited in this specification are herein incorporated byreference as if each was specifically and individually indicated to beincorporated by reference.

ADDITIONAL SEQUENCE INFORMATION

SEQ ID NO: 1. Human PAR1 1 MGPRRLLLVA ACFSLCGPLL SARTRARRPE SKATNATLDP41 RSFLLRNPND KYEPFWEDEE KNESGLTEYR LVSINKSSPL 81QKQLPAFISE DASGYLTSSW LTLFVPSVYT GVFVVSLPLN 121IMAIVVFILK MKVKKPAVVY MLHLATADVL FVSVLPFKIS 161YYFSGSDWQF GSELCRFVTA AFYCNMYASI LLMTVISIDR 201FLAVVYPMQS LSWRTLGRAS FTCLAIWALA IAGVVPLLLK 241EQTIQVPGLN ITTCHDVLNE TLLEGYYAYY FSAFSAVFFF 281VPLIISTVCY VSIIRCLSSS AVANRSKKSR ALFLSAAVFC IFIICFGPTN 331VLLIAHYSFL SHTSTTEAAY FAYLLCVCVS SISCCIDPLI 371YYYASSECQR YVYSILCCKE SSDPSSYNSS GQLMASKMDT 411 CSSNLNNSIY KKLLTSEQ ID NO: 2. Human PAR1 Met¹-Arg⁴⁶ deleted fragment 47 NPND KYEPFWEDEE61 KNESGLTEYR LVSINKSSPL QKQLPAFISE DASGYLTSSW LTLFVPSVYT 111GVFVVSLPLN IMAIVVFILK MKVKKPAVVY MLHLATADVL 151FVSVLPFKIS YYFSGSDWQF GSELCRFVTA AFYCNMYASI 191LLMTVISIDR FLAVVYPMQS LSWRTLGRAS FTCLAIWALA 231IAGVVPLLLK EQTIQVPGLN ITTCHDVLNE TLLEGYYAYY 271FSAFSAVFFF VPLIISTVCY VSIIRCLSSS AVANRSKKSR ALFLSAAVFC 321IFIICFGPTN VLLIAHYSFL SHTSTTEAAY FAYLLCVCVS SISCCIDPLI 371YYYASSECQR YVYSILCCKE SSDPSSYNSS GQLMASKMDT 411 CSSNLNNSIY KKLLTSEQ ID NO: 3. Human PAR1 Asn⁴⁷-Trp¹⁰⁰ fragmentNPND KYEPFWEDEE KNESGLTEYR LVSINKSSPL  QKQLPAFISE DASGYLTSSW

What is claimed is:
 1. A polypeptide derived from protease activatedreceptor-1 (PAR1), consisting of an N-terminal fragment of human PAR1extracellular region Asn⁴⁷-Trp¹⁰⁰ (SEQ ID NO:3) or conservativelymodified variant thereof, wherein the fragment comprises at least thefirst 22 N-terminal residues of SEQ ID NO:3.
 2. The polypeptide of claim1, wherein the fragment comprises at least the first 25 N-terminalresidues of human PAR1 extracellular fragment Asn⁴⁷-Trp¹⁰⁰ (SEQ IDNO:3).
 3. The polypeptide of claim 1, wherein the fragment comprises atleast the first 25 N-terminal residues that are identical to thecorresponding N-terminal residues of human PAR1 extracellular regionAsn⁴⁷-Trp¹⁰⁰ (SEQ ID NO:3).
 4. The polypeptide of claim 1, consisting ofan amino acid sequence shown in SEQ ID NO:3.
 5. A peptide derived fromprotease activated receptor-1 (PAR1), consisting of 10, 11, 12, 13, 14,16, 17, 18, 19 or 20 amino acid residues that are identical to thecorresponding N-terminal residues of human PAR1 extracellular regionAsn⁴⁷-Trp¹⁰⁰ (SEQ ID NO:3) or conservatively modified variant thereof.6. The polypeptide of claim 5, consisting of an amino acid sequence asshown in any one of SEQ ID NOs:4-7.
 7. An isolated polynucleotideencoding the polypeptide of claim
 1. 8. A method of promotingcytoprotective activity for endothelial cells, comprising contacting thecells with a PAR1-derived cytoprotective polypeptide, wherein thePAR1-derived polypeptide (1) consisting of an N-terminal fragment ofhuman PAR1 extracellular fragment Asn⁴⁷-Trp¹⁰⁰ (SEQ ID NO:3) orconservatively modified variant thereof, wherein the fragment comprisesat least the first 22 N-terminal residues of SEQ ID NO:3 or (2) consistsof an amino acid sequence that is identical to the first 6, 7, 8, 10,11, 12, 13, 14, 16, 17, 18, 19 or 20 N-terminal residues of human PAR1extracellular fragment Asn⁴⁷-Trp¹⁰⁰ (SEQ ID NO:3) or conservativelymodified variant thereof.
 9. The method of claim 8, wherein thepolypeptide consists of an amino acid sequence as shown in SEQ ID NO:4(NPNDKYEPFWEDEEKNESGL).
 10. The method of claim 8, wherein theendothelial cells are present in a subject.
 11. The method of claim 8,wherein the polypeptide is administered to a subject to reduce mortalityfrom adult severe sepsis or pediatric meningococcemia; to promote woundhealing in diabetic ulcer; to treat injuries from ischemic stroke,neurotrauma, or other acute or chronic neurodegenerative conditions; totreat injuries from cardiac ischemia/reperfusion, hepaticischemia/reperfusion, renal ischemia/reperfusion; to treat inflammatorylung injury or gastrointestinal injury; to treat flap necrosis inreconstructive surgery; to prolong survival following Ebola infection;or to reduce injury caused by radiation.
 12. The polypeptide of claim 5,consisting of an amino acid sequence as shown in SEQ ID NO:4(NPNDKYEPFWEDEEKNESGL) or conservatively modified variant thereof.
 13. Asynthetic or isolated peptide derived from protease activated receptor-1(PAR1), consisting of an amino acid sequence that is identical to thefirst 6, 7, or 8 N-terminal residues of the Met¹-Arg⁴⁶ deleted humanPAR1 sequence (SEQ ID NO:2) or conservatively modified variant thereof.14. The peptide of claim 13, consisting of NPNDKY (SEQ ID NO:14) orconservatively modified variant thereof.
 15. The polypeptide of claim 5,consisting of an amino acid sequence as shown in NPNDKYEPFW (SEQ IDNO:16), NPNDKYEPFWED (SEQ ID NO:17), NPNDKYEPFWEDEE (SEQ ID NO:18),NPNDKYEPFWEDEEKN (SEQ ID NO:19) or NPNDKYEPFWEDEEKNES (SEQ ID NO:20).16. The peptide of claim 13, consisting of NPNDKYEP (SEQ ID NO:15) orconservatively modified variant thereof.